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The effects of hydrodynamics and organic matter on sediment biofilm esterase activity and [H-3]thymidine incorporation were investigated at several depths in the streambed and riparian zone of the alpine stream Oberer Seebach (Austria). On the sediment scale, microbial activity increased with both upwelling and downwelling Darcian velocities of interstitial water and was minimal when the surface/subsurface water exchange decreased. As revealed by stepwise multiple regression analyses, sediment carbohydrates and chlorophyll a explained a considerable percentage (up to 97% in summer) of the variance of biofilm esterase activity and [H-3]thymidine incorporation, whereas porewater temperature and concentrations of dissolved organic carbon (DOC) and dissolved oxygen (DO) were poor predictors of biofilm activity. DOC and DO fluxes, however, accounted for a considerable percentage of the variance in [H-3]thymidine incorporation, which points toward the importance of how in biofilm functioning. On the reach scale, the ratio of surface water travel length to the subsurface travel length, which is an index of the surface/subsurface water exchange frequency, determined average reach biofilm activity. Downstream routing along predominantly subsurface flow paths increased streambed esterase activity, whereas elevated surface travel lengths reduced esterase activity. Esterase activity and [H-3]thymidine incorporation were also positively related to the streambed DOC retention efficiency, which underscores the role of biofilms in solute retention. These results highlight the coupling between streambed hydrodynamics and microbial activity and link sediment-scale and reach-scale processes.