We used in situ nutrient-enrichment experiments at sites in 4 Texas streams spanning a steep nutrient gradient to test the hypothesis that periphyton photoautotrophs and heterotrophs are differentially limited by N and P, respectively. We contrasted the responses of total periphyton biomass (ash-free dry mass [AFDM]), photoautotrophic biomass (chlorophyll a [CHLA]), % CHLA of AFDM [% CHLA]), alkaline phosphatase activity (APA), and nitrogenase activity (NA) to ambient N and P concentrations among sites and among nutrient-diffusing substrata (NDS) (control, N, P, and N P) within sites. Periphyton biomass (AFDM and CHLA) increased steadily with increasing ambient N and P among sites. Both APA and NA were relatively high on the control treatments at the lowest nutrient site and decreased with increasing ambient N and P until falling to negligible levels at the highest nutrient site. Within sites, P enrichment decreased APA but did not increase % CHLA or CHLA/cm². P enrichment did increase AFDM/cm² at one of the low nutrient sites. N enrichment reduced NA and increased % CHLA and CHLA/cm² without increasing AFDM/cm². N P enrichment resulted in the highest % CHLA and CHLA/cm² at the 3 lowest nutrient sites but did not increase AFDM/cm². Combined, these findings suggest that photoautotrophs in periphyton communities may be more limited than heterotrophs by N or become more competitive for P under elevated N. The high levels of APA and NA on the controls coupled with minimal difference in AFDM among nutrient treatments at most sites despite increases in % CHLA on N and N P treatments provide experimental evidence that differential nutrient limitation probably occurs in periphyton communities. Our results support the view that the single-limiting-nutrient paradigm may lead to overly simplistic interpretations of periphyton responses to nutrient enrichment.