We explored N₂ fixation and alkaline phosphatase activity (APA) in periphyton from a N-limited stream ecosystem by coupling measurements of these processes with nutrient diffusion substrata (NDS) experiments. We measured periphyton biomass accumulation (as ash-free dry mass [AFDM] and chlorophyll a [CHLA]), N₂ fixation, and APA to evaluate the relative importance of N₂ fixation as an N source to the periphyton community and APA as an indicator of P deficiency in a seemingly N-limited system. We used fritted-glass-disc NDS and estimated AFDM, CHLA, N₂ fixation, and APA on days 6, 18, and 29 after deployment. Periphyton AFDM steadily increased on NDS over time, but was not influenced by nutrients. CHLA was elevated in the N treatment on days 18 and 29, indicating autotrophic N limitation. Consistent with N limitation, N₂ fixation was high but not different in the control and P treatments and was virtually undetectable on the N treatment. N₂ fixation in control and P treatments was detectable in both light and dark incubations, and dark rates were 4 to 73% of the light rates on days 18 and 29. The average contribution of total N₂ fixation to periphyton in control and P treatments was 0.93 mg N/m² on day 18 and 1.0 mg N/m² on day 29. APA was significantly elevated on the control and was highest in the N treatment despite no apparent P limitation of periphyton biomass accumulation. P enrichment always decreased APA. Measurable N₂ fixation and the change in CHLA suggest that autotrophs were primarily N limited. However, APA observed in controls demonstrated that some portion of the periphyton community was experiencing P deficiency. This result suggests that periphyton metabolism was related to both N and P availability, but that biomass accumulation might have been limited primarily by N. One explanation for these findings is that different organisms, perhaps occupying different trophic positions within the community, might have been limited by different elements.