The U.S. Geological Survey, in cooperation with the Massachusetts Department of Conservation and Recreation, Massachusetts Department of Environmental Protection, and the Massachusetts Department of Fish and Game, conducted an investigation of fish assemblages in small- to medium-sized Massachusetts streams. The objective of this study was to determine relations between fish-assemblage characteristics and anthropogenic factors, including impervious cover and estimated flow alteration, relative to the effects of environmental factors, including physical-basin characteristics and land use. The results of this investigation supersede those of a preliminary analysis published in 2010. Fish data were obtained for 669 fish-sampling sites from the Massachusetts Division of Fisheries and Wildlife fish-community database. A review of the literature was used to select fish metrics - species richness, abundance of individual species, and abundances of species grouped on life history traits - responsive to flow alteration. The contributing areas to the fish-sampling sites were delineated and used with a geographic information system to determine a set of environmental and anthropogenic factors that were tested for use as explanatory variables in regression models. Reported and estimated withdrawals and return flows were used together with simulated unaltered streamflows to estimate altered streamflows and indicators of flow alteration for each fish-sampling site. Altered streamflows and indicators of flow alteration were calculated on the basis of methods developed in a previous U.S. Geological Survey study in which unaltered daily streamflows were simulated for a 44-year period (water years 1961-2004), and streamflow alterations were estimated by use of water-withdrawal and wastewater-return data previously reported to the State for the 2000-04 period and estimated domestic-well withdrawals and septic-system discharges. A variable selection process, conducted using principal components analysis and Spearman rank correlation, was used to select a set of 15 non-redundant environmental and anthropogenic factors to test for use as explanatory variables in the regression analyses. Twenty-one fish species were used in a multivariate analysis of fish-assemblage patterns. Results of nonmetric multidimensional scaling and hierarchical cluster analysis were used to group fish species into fluvial and macrohabitat generalist habitat-use classes. Two analytical techniques, quantile regression and generalized linear modeling, were applied to characterize the association between fish-response variables and environmental and anthropogenic explanatory variables. Quantile regression demonstrated that as percent impervious cover and an indicator of percent alteration of August median flow from groundwater withdrawals increase, the relative abundance and species richness of fluvial fish decrease. The quantile regression plots indicate that (1) as many as seven fluvial fish species are expected in streams with little flow alteration or impervious cover, (2) no more than four fluvial fish species are expected in streams where flow alterations from groundwater withdrawals exceed 50 percent of the August median flow or the percent area of impervious cover exceeds 15 percent, and (3) few fluvial fish remain at high rates of withdrawal (approaching 100 percent) or high rates of impervious cover (between 25 and 30 percent). Three generalized linear models (GLMs) were developed to quantify the response of fluvial fish to multiple environmental and anthropogenic variables. All variables in the GLM equations were demonstrated to be significant (p less than 0.05, with most less than 0.01). Variables in the fluvial-fish relative-abundance model were channel slope, estimated percent alteration of August median flow from groundwater withdrawals, percent wetland in a 240-meter buffer strip, and percent impervious cover. Variables in the fluvial-fish species-richness model were drainage area, channel slope, total undammed reach length, percent wetland in a 240-meter buffer strip, and percent impervious cover. Variables in the brook trout relativeabundance model were drainage area, percent open water, and percent impervious cover. The variability explained by the GLM models, as measured by the pseudo R2, ranged from 18.2 to 34.6, and correlations between observed and predicted values ranged from 0.50 to 0.60. Results of GLM models indicated that, keeping all other variables the same, a one-unit (1 percent) increase in the percent depletion of August median flow would result in a 0.9-percent decrease in the relative abundance (in counts per hour) of fluvial fish. The results of GLM models also indicated that a unit increase in impervious cover (1 percent) resulted in a 3.7-percent decrease in the relative abundance of fluvial fish, a 5.4-percent decrease in fluvial-fish species richness, and an 8.7-percent decrease in brook trout relative abundance.