Nitrous oxide (N2O), a trace gas whose concentration is increasing in the atmosphere, plays an important role in both radiative forcing and stratospheric ozone depletion(1,2). Its biogeochemical cycle has thus come under intense scrutiny in recent years. Despite these efforts, the global budget of N2O remains unresolved, and the nature and magnitude of the sources and sinks continue to be debated(3-5) despite the constraints that can be provided by characterizations of the gas(6,7). We report here the results of dual-isotope measurements of N2O from the water column of the subtropical North Pacific Ocean. Nitrous oxide within the lower-euphotic and upper-aphotic zones is depleted in both N-15 and O-18 relative to its tropospheric and deep-ocean composition. These findings are consistent with a prediction, based on global mass-balance considerations, of a near-surface isotopically depleted oceanic N2O source(4). Our results indicate that this source, probably produced by bacterial nitrification, contributes significantly to the ocean-atmosphere flux of N2O in the oligotrophic subtropical North Pacific Ocean. This source may act to buffer the isotopic composition of tropospheric N2O and is quantitatively significant in the global tropospheric N2O budget. Because dissolved gases in near-surface waters are more readily exchanged with the atmospheric reservoir than those in deep waters, the existence of a quantitatively significant N2O source at a relatively shallow depth has potentially important implications for the susceptibility of the source, and the ocean-atmosphere flux, to climatic influences.