The Santa Ana River basin is the largest stream system in Southern California and includes a densely populated coastal area. Extensive urbanization has altered the geomorphology and hydrology of the streams, adversely affecting aquatic communities. We studied macroinvertebrate and periphyton assemblages in relation to two categorical features of the highly engineered hydrologic system-water source and channel type. Four water sources were identified-natural, urban-impacted groundwater, urban runoff, and treated wastewater. Three channel types were identified-natural, channelized with natural bottom, and concrete-lined. Nineteen sites, covering the range of these two categorical features, were sampled in summer 2000. To minimize the effects of different substrate types among sites, artificial substrates were used for assessing macroinvertebrate and periphyton assemblages. Physical and chemical variables and metrics calculated from macroinvertebrate and periphyton assemblage data were compared among water sources and channel types using analysis of variance and multiple comparison tests. Macroinvertebrate metrics exhibiting significant (P < 0.05) differences between water sources included taxa and Ephemeroptera-Plecoptera-Trichoptera richness, relative richness and abundance of nonchironomid dipterans, orthoclads, oligochaetes, and some functional-feeding groups such as parasites and shredders. Periphyton metrics showing significant differences between water sources included blue-green algae biovolume and relative abundance of nitrogen heterotrophic, eutrophic, motile, and pollution-sensitive diatoms. The relative abundance of trichopterans, tanytarsini chironomids, noninsects, and filter feeders, as well as the relative richness and abundance of diatoms, were significantly different between channel types. Most physical variables were related to channel type, whereas chemical variables and some physical variables (e.g., discharge, velocity, and channel width) were related to water source. These associations were reflected in correlations between metrics, chemical variables, and physical variables. Significant improvements in the aquatic ecosystem of the Santa Ana River basin are possible with management actions such as conversion of concrete-lined channels to channelized streams with natural bottoms that can still maintain flood control to protect life and property.