We examined the role of nutrients in structuring algal periphyton communities in 12 softwater lakes varying widely in acidity by a) evaluating species composition on unenriched substrata in relation to water chemistry, and b) measuring the growth responses of dominant species within the communities of each lake to enrichment with eight combinations of N, P, and C. Species composition on substrata deployed for 5-6 wk in each lake was strongly related to pH and alkalinity, and relatively unresponsive to differences in either dissolved inorganic N (DIN) or total P in the water column, as determined using Canonical Correspondence Analysis. The limiting nutrient for at least some species, determined from their growth responses to nutrient enrichment, was in contrast closely related to water column nutrients. Low ratios of dissolved inorganic C (DIC) to DIN in the most acidic lakes were associated with an increase in the number of C-limited species, and with shifts from N limitation to C limitation of a few prevalent taxa. The N-fixing cyanophyte Nostoc commune was one of very few species to be frequently limited by the same nutrient (P) regardless of lake type. Although at least a portion of the periphyton community is clearly nutrient limited in these lakes during midsummer, moderate variation in the concentrations of water-column nutrients may be insufficient to promote large-scale changes in species composition because of the considerable attenuation of nutrient availability to cells within the periphyton mat.