Spatial and temporal variations in stable carbon isotope ratios (i.e., δ13C) of primary producers are common but poorly understood features of isotopic characterizations of aquatic food webs. I investigated factors that control δ13C of algae (concentration and δ13C of inorganic carbon, algal fractionation, and growth rates) in riffle habitats across a gradient in stream size and productivity in northern California. There was considerable seasonal and spatial variation in δ13C of the green alga Cladophora glomerata, microalgal-influenced epilithic biofilms, and their herbivores. Algal and herbivore δ13C were depleted in 13C in small, unproductive tributary streams (-44‰ to -30‰) compared with more productive sites downstream (-31‰ to -23‰). The majority of variation in algal δ13C of Cladophora and epilithic biofilms was determined by dissolved CO2 (CO2aq,) via effects on δ13C of CO2aq and photosynthetic fractionation. In contrast, two other taxa (the cyanobacterium Nostoc pruniforme and the red alga Lemanea sp.) showed little variation in δ13C or fractionation in response to varied inorganic carbon availability because of their distinct modes of inorganic carbon acquisition. Although variation in algal δ13C might complicate use of δ13C to resolve consumer diet sources under some circumstances, better understanding of such variation should improve the use of δ13C techniques in aquatic food web studies.