We assessed the structure of periphyton, benthic macroinvertebrate, and fish assemblages and their associations with environmental variables at 17 sites on streams of the highly urbanized Santa Ana River basin in Southern California. All assemblages exhibited strong differences between highly urbanized sites in the valley and the least-impacted sites at the transition between the valley and undeveloped mountains. Results within the urbanized area differed among taxa. Periphyton assemblages were dominated by diatoms (>75% of total taxa). Periphyton assemblages within the urbanized area were not associated with any of the measured environmental variables, suggesting that structure of urban periphyton assemblages might be highly dependent on colonization dynamics. The number of Ephemeroptera, Trichoptera, and Plecoptera (EPT) taxa included in macroinvertebrate assemblages ranged from 0 to 6 at urbanized sites. Benthic macroinvertebrate assemblages had significant correlations with several environmental variables within the urban area, suggesting that stream size and permanence were important determinants of distribution among the species able to survive conditions in urban streams. Only 4 of 16 fish species collected were native to the drainage. Fish assemblages of urbanized sites included two native species, arroyo chub Gila orcuttii and Santa Ana sucker Catostomus santaanae, at sites that were intermediate in coefficient of variation of bank-full width, depth, bed substrate, and water temperature. Alien species dominated urbanized sites with lesser or greater values for these variables. These results suggest that urban streams can be structured to enhance populations of native fishes. Continued study of urban streams in the Santa Ana River basin and elsewhere will contribute to the basic understanding of ecological principles and help preserve the maximum ecological value of streams in highly urbanized areas.