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Background aqueous chemistry and ¹⁵N_nitrate tracer injection methods were used to calculate in‐stream nitrate uptake metrics at Red Canyon Creek, a third‐order stream in the Rocky Mountains in the state of Wyoming, United States. ‘Net’ nitrate uptake lengths, which reflect both nitrate uptake and regeneration, and ‘gross’ nitrate uptake lengths, which exclude re‐mineralization, were quantified separately from background nitrate chemistry and ¹⁵N labelling tracer data, respectively. Gross nitrate uptake lengths, from tracer injections of ¹⁵N labelled nitrate, ranged from 502 to 3140 m. Net nitrate uptake lengths, from background nitrate chemistry downstream of a point source, ranged from 1170 to 4330 m. Diurnal changes in uptake lengths suggest the importance of nitrate utilization by autotrophs in the stream and benthic zone. The differences between net and gross nitrate uptake lengths along lower reaches of …