The major ion chemistry of water from an 11.42-km reach of the Pajaro River, a losing stream in central coastal California, shows a consistent pattern of higher concentrations during the 2nd (dry) half of the water year. Most solutes are conserved during flow along the reach, but [NO3−] decreases by ~30% and is accompanied by net loss of channel discharge and extensive surface–subsurface exchange. The corresponding net NO3− uptake length is 37 ± 13 km (42 ± 12 km when normalized to the conservative solute Cl−), and the areal NO3− uptake rate is 0.5 μmol m−2 s−1. The observed reduction in [NO3−] along the reach results from one or more internal sinks, not dilution by ground water, hill-slope water, or other water inputs. Observed reductions in [NO3−] and channel discharge along the experimental reach result in a net loss of 200–400 kg/d of NO3−-N, ~50% of the input load. High-resolution (temporal and spatial) sampling indicates that most of the NO3− loss occurs along the lower part of the reach, where there is the greatest seepage loss and surface–subsurface exchange of water. Stable isotopes of NO3−, total dissolved P concentrations, and streambed chemical profiles suggest that denitrification is the most significant NO3− sink along the reach. Denitrification efficiency, as expressed through downstream enrichment in 15N-NO3−, varies considerably during the water year. When discharge is greater (typically earlier in the water year), denitrification is least efficient and downstream enrichment in 15N-NO3− is greatest. When discharge is lower, denitrification in the streambed appears to occur with greater efficiency, resulting in lower downstream enrichment in 15N-NO3−.