The San Joaquin River (SJR) in California is purported to receive high nitrate loadings from surrounding agricultural lands through both surface and groundwater inputs. To investigate the potential removal of nitrate (NO3-) from surface and ground water sources, the spatial variations in dinitrogen (N2) gas concentrations and direct measurements of sediment denitrification potential (DNP), with amended NO3- and carbon (C) treatments, were investigated in the summer along a 95-km reach of the San Joaquin River. Excess N2 in hyporheic porewaters ranged from <0.1 to 8.65 mg L-1 and was significantly higher in porewaters from the 1.3 m (ground water source) versus 0.3 m (mixed surface and ground water) depths. In deep groundwater wells (3-7 m), median excess N2 concentration was 5.39 mg L-1 (range = <0.1-14.6 mg L-1). Excess N2 concentrations were inversely correlated with dissolved oxygen and NO3- concentrations suggesting denitrification as an important process in the dominantly anaerobic sediments. Hyporheic porewater NO3- concentrations exceeded the detection limit of 0.01 mg L-1 in only 20% of the hyporheic porewaters, in spite of high NO3- concentrations measured in both surface waters (mean = 2.25 mg N L-1) and surrounding groundwaters. Sediment DNP rates averaged 253 and 297 μg N kg-1 hr-1 for NO3- amended, and NO3- + C amended sediments, respectively, supporting the prevalence of denitrification in hyporheic sediments. Our results indicate that the hyporheic/riparian zones act as an anoxic barrier to nitrate transport from regional groundwater and as a location to remove NO3- from surface waters exchanging with the hyporheic zone.