The effects of acidification in lotic systems are not well documented. Spatial and temporal variability of habitat and water quality complicate the evaluation of acidification effects in streams and rivers. The Neversink River in the Catskill Mountains of southeastern New York, the tributaries of which vary from well buffered to severely acidified, provided an opportunity to investigate the extent and magnitude of acidification effects on fish communities of headwater systems. Composition of fish communities, water quality, stream hydrology, stream habitat, and physiographic factors were characterized from 1991 to 1995 at 16 first- to fourth-order sites in the basin. Correlation and regression analyses were used to develop empirical models and to assess the relations among fish species richness, total fish density, and total fish biomass and environmental variables. Chronic and episodic acidification and elevated concentrations of inorganic monomeric aluminum were common, and fish populations were rare or absent from several sites in the upper reaches of the basin; as many as six fish species were collected from sites in the lower reaches of the basin. Species distributions and species richness were most highly related to stream pH, acid-neutralizing capacity (ANC), inorganic monomeric aluminum (Al_im), calcium (Ca)²⁺, and potassium (K)⁺ concentrations, site elevation, watershed drainage area, and water temperature. Fish density was most highly related to stream pH, Al_im, ANC, K⁺, Ca²⁺, and magnesium (Mg)²⁺ concentrations. Fish biomass, unlike species richness and fish density, was most highly related to physical habitat characteristics, water temperature, and concentrations of Mg²⁺ and silicon. Acidity characteristics were of secondary importance to fish biomass at all but the most severely acidified sites. Our results indicate that (1) the total biomass of fish communities was not seriously affected at moderately to strongly acidified sites; (2) species richness and total density of fish were adversely affected at strongly to severely acidified sites; and (3) possible changes in competitive interactions may mitigate negative effects of acidification on fish communities in parts of the Neversink River Basin.