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Stream networks can retain or remove nutrient pollution, including nitrate from agricultural and urban runoff. However, assessing the location and timing of nutrient uptake remains challenging because of the hydrological and biogeochemical complexity of dynamic stream ecosystems. We used a novel approach to continuously characterize the biological activity in a stream with in situ measurement of dissolved gases by membrane inlet mass spectrometry (MIMS). In a headwater stream in western France, we compared in situ measurements of O2, CO2, N2, and N2O (the main gases associated with respiration, including denitrification) with more traditional laboratory incubations of collected sediment. The in situ measurements showed near-zero denitrification in the stream and the hyporheic zone. However, the laboratory incubations showed a low but present denitrification potential. This demonstrates how denitrification potential is not necessarily expressed in field hydrological and geochemical conditions. In situ measurements are thus crucial to quantify expressed rates of nutrient removal. Broader application of in situ gas measurement based on technologies such as MIMS could enhance our understanding of the spatiotemporal distribution of stream and hyporheic processes and overall nutrient retention at stream network scales.