The priming effect refers to quantitative changes in microbial decomposition of recalcitrant organic matter upon addition of labile organic matter and is a phenomenon that mainly has been reported and debated in soil science. Recently, priming effects have been indicated in aquatic ecosystems and have received attention due to the potential significance for ecosystem carbon budgets. Headwater stream biofilms, which are important degraders of both allochthonous, presumably recalcitrant, organic matter and labile autochthonous organic matter, may be sites where priming effects are important in aquatic environments. We have experimentally tested for priming effects in stream biofilms within microcosms mimicking the stream hyporheic zone. A 13C labeled model allochthonous carbon source was used in combination with different carbon sources simulating autochthonous inputs. We did not detect changes in respiration, removal or incorporation of allochthonous organic matter in response to autochthonous treatments, thus not supporting the occurrence of priming effects under the experimental conditions. This study is the first to address priming effects in the hyporheic zone, and one of very few studies quantitatively assessing aquatic priming effects. The results contrast with existing studies, which highlights the need for quantitative approaches to determine the importance of priming effects in aquatic environments.