Rainbow trout Oncorhynchus mykiss have been widely stocked throughout the United States as a popular sport fish. Our study was initiated to evaluate potential effects of rainbow trout introduction on native fishes to inform future decisions about trout stocking in northeastern Oklahoma streams. We sampled fish assemblages in pools, glides, and riffles in Brush Creek, Delaware County, Oklahoma, from February 2000 to September 2002, and experimentally stocked rainbow trout into the stream from November 2000 to March 2001 and November 2001 to March 2002. We used a combination of multivariate analyses to evaluate seasonal and habitat effects on native fish assemblages and to compare assemblage structure between prestocking, the first year of stocking, and the second year of stocking. Mesohabitat type significantly affected assemblage structure among years, whereas we did not detect an effect of season. We did not detect differences in assemblage structure among years in glide or riffle habitats. Native fish assemblage structure in pool habitats before rainbow trout introduction differed from assemblage structure in both the first and second year of stocking. Declines in seven species, including two native game fish (smallmouth bass Micropterus dolomieu and bluegill Lepomis machrochirus), contributed to assemblage dissimilarity in pool habitats between prestocking conditions and the second year of stocking. Our results indicate that stocking rainbow trout may cause local disruption in assemblage structure in pool habitats. ?? 2004 by the American Fisheries Society.