The fishes in Lower Putah Creek, a regulated stream in the Central Valley of California, were sampled over a 5-yr period, 1994-1998. Distinct fish assemblages were observed in the lower 37 km of stream using two-way indicator species analysis (TWIN-SPAN) and canonical correspondence analysis (CCA). The assemblages segregated in an upstream-to-downstream manner. Distinct differences were found between assemblages of native and nonnative fishes and their association with environmental variables and habitat use. Native fishes tended to cluster in areas with colder temperatures, lower conductivity, less pool habitat, faster streamflow, and more shaded stream surface. Numbers of nonnative fish were negatively correlated with increased streamflow, and numbers of native fish were positively correlated with increased flow. Hydrologic variability between years and seasons indicated that flow regime had a large effect on the fish assemblages. This study provides a clear demonstration of how native fishes in streams of the western United States exhibit different habitat requirements and respond to temporal variation in flow in a different manner than nonnative fishes. It supports the concept that restoration of natural flow regimes, in company with other restoration measures, is necessary if the continued downward decline of native fish populations in the western United States is to be reversed.