The idea that small amounts of labile organic carbon might trigger the degradation of previously unreactive organic matter has attracted increasing scientific interest across multiple disciplines. Although this phenomenon, referred to as priming, has been widely reported in soils, evidence in freshwater systems is scarce and inconclusive. Here, we use a multifactorial microcosm experiment to test the conditions under which priming may be observed in freshwater ecosystems. We assessed the effect of pulse additions of three labile carbon sources (acetate, glucose, and cellobiose) on dissolved organic carbon (DOC) consumption using water from lakes with different trophic states (eutrophic to oligotrophic and clear to brownwater lakes). We further analyzed the effect of nutrient availability and the role of attachment of cells to surfaces. Despite the range of conditions tested, we found no clear evidence of a priming effect on DOC degradation, indicating that priming in freshwater systems may be of limited importance.