ABSTRACT. – Stable carbon isotopes are increasingly used to estimate the relative contribution of phytoplankton and periphyton towards secondary production in aquatic food webs. This approach, developed mostly through studies of deep stratified lakes, remains to be evaluated in the context of large rivers, where factors such as short water residence time, little stratification, and variable turbidity may affect carbon fractionation by primary producers. We used invertebrate filterers and grazers as integrators of isotopic ratios of phytoplankton and periphyton, respectively, in twelve stations from a large (35 km) fluvial lake of the Saint-Lawrence River, Québec, Canada. We found strong but variable differences in Δδ¹³C, ranging from 1 to 7 ‰, between grazers and filterers collected at the same sites. Following an experimental approach we then showed that the light regime, known for influencing isotopic fractionation from dissolved inorganic carbon, accounted for very little variability in the isotopic ratio of periphyton. On the other hand, field data showed that the isotopic difference between filterers and grazers was strongly related to spatial variation in the relative abundance of phytoplankton and periphyton within the lake. This study showed that the basis of benthic food web production in a fluvial lake is spatially highly variable and can be efficiently quantified with carbon isotopes. Our results revealed the existence of a complex spatial mosaic ranging from an almost complete trophic dependence on phytoplankton to an important assimilation of non-sestonic sources.