AbstractLarge variability in dissolved organic carbon (DOC) uptake rates has been reported for headwater streams, but the causes of this variability are still not well understood. Here we assessed acetate uptake rates across 11 European streams comprising different ecoregions by using whole‐reach pulse acetate additions. We evaluated the main climatic and biogeochemical drivers of acetate uptake during two seasonal periods. Our results show a minor influence of sampling periods but a strong effect of climate and dissolved organic matter (DOM) composition on acetate uptake. In particular, mean annual precipitation explained half of the variability of the acetate uptake velocities (VfAcetate) across streams. Temperate streams presented the lowestVfAcetate, together with humic‐like DOM and the highest stream respiration rates. In contrast, higherVfAcetatewere found in semiarid streams, with protein‐like DOM, indicating a dominance of reactive, labile compounds. This, together with lower stream respiration rates and molar ratios of DOC to nitrate, suggests a strong C limitation in semiarid streams, likely due to reduced inputs from the catchment. Overall, this study highlights the interplay of climate and DOM composition and its relevance to understand the biogeochemical mechanisms controlling DOC uptake in streams.