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Benthic biofilms have multiple functions in stream and river ecosystems, and their growth and productivity are often limited by dissolved inorganic nutrient availability, particularly N or P. We deployed nutrient diffusing substrata (NDS) in 5 rivers in each of 3 regions: the Mountain West, the Arid West, and the Midwest, to assess regional and seasonal variation in nutrient limitation of river biofilms and to quantify which environmental factors control nutrient limitation. We constructed NDS with either inorganic or organic substrata to compare nutrient limitation of autotrophic and heterotrophic biofilms, and we explored inorganic N preference of biofilms by comparing results of NO3- and NH4+ amendments. During summer deployments, autotrophic and heterotrophic biofilms in Mountain Western rivers were commonly limited by N or colimited by N and P, whereas nutrient limitation was less common in Arid Western and Midwestern rivers. We deployed NDS seasonally in Midwestern rivers and found no obvious seasonal patterns of nutrient limitation, probably because background nutrient concentrations were consistently high. Heterotrophic biofilm responses in Midwestern rivers suggested a preference for NO3- over NH4+. The magnitude of the autotrophic biofilm response, calculated as response ratios for gross primary production and chlorophyll a, were inversely related to % developed lands in the surrounding watershed and background NO3- availability, whereas heterotrophic response ratios were related to river temperature. Patterns of nutrient limitation for autotrophic biofilms in rivers were similar to those in headwater streams, suggesting that biofilms in lotic ecosystems of varying size are similarly sensitive to nutrient amendments from the landscape. Clarifying the nutrient-limitation status of autotrophic and heterotrophic biofilms across a variety of rivers expands our understanding of nutrient limitation in lotic ecosystems and enables improved management of autotrophic and heterotrophic biofilms.