We investigated factors that contribute to isotopic carbon fractionation in periphytic biofilms in a human-altered headwater stream with a flashy hydrograph. Water velocity had an important effect on periphyton d 13 C, explaining both temporal and spatial variation. We found that water velocity averaged over a certain period before sampling, rather than the instantaneous water velocity, explained a high percentage of both temporal and spatial variation of the periphyton d 13 C signature. The relationship between water velocity and periphyton d 13 C signature was particu- larly influenced by individual flow events during the recent flow history. A simple model based on a flow history of 3-4 weeks reliably estimated the d 13 C signature of periphyton from distinct reaches. The model clearly identified signature shifts caused by the deposition of activated sludge particles from a wastewater treatment plant onto the periphytic biofilms. We highlight the high spatial and temporal variability of periphyton d 13 C signatures (i.e., up to 3-6‰) in a heterogeneous flow environment with inputs from a wastewater treatment plant, and we explore its implications for food web analysis.