We used an in situ steady state ¹⁵N-labeled nitrate (¹⁵NO₃⁻) and acetate (AcO⁻) well-to-wells injection experiment to determine how the availability of labile dissolved organic carbon (DOC) as AcO⁻ influences microbial denitrification in the hyporheic zone of an upland (third-order) agricultural stream. The experimental wells receiving conservative (Cl⁻ and Br) and reactive (¹⁵NO₃⁻) solute tracers had hyporheic median residence times of 7.0 to 13.1 h, nominal flowpath lengths of 0.7 to 3.7 m, and hypoxic conditions (<1.5 mg O₂ L⁻¹). All receiving wells demonstrated ¹⁵N₂ production during ambient conditions, indicating that the hyporheic zone was an environment with active denitrification. The subsequent addition of AcO⁻ stimulated more denitrification as evidenced by significant δ¹⁵N₂ increases by factors of 2.7 to 26.1 in receiving wells and significant decreases of NO₃⁻ and DO in the two wells most hydrologically connected to the injection. The rate of nitrate removal in the hyporheic zone increased from 218 kg ha⁻¹ yr⁻¹ to 521 kg ha⁻¹ yr⁻¹ under elevated AcO⁻ conditions. In all receiving wells, increases of bromide and ¹⁵N₂ occurred without concurrent increases in AcO⁻, indicating that 100% of AcO⁻ was retained or lost in the hyporheic zone. These results support the hypothesis that denitrification in anaerobic portions of the hyporheic zone is limited by labile DOC supply
