A relatively narrow vertical zone (5–6 m thick) of NO₃⁻ containing groundwater was identified using multilevel sampling devices in a sand and gravel aquifer on Cape Cod, MA, USA. The aquifer has been chronically contaminated by surface disposal of treated sewage 0.3 km upgradient from the study area. The NO₃⁻ zone was anoxic and contained high concentrations of N₂O (16.5 μM), suggesting that it was a zone of active denitrification. Denitrifying activity was confirmed with direct measurement using acetylene block incubations with aquifer core material; the peak rate was 2.4 nmol N reduced (g sed)⁻¹ day⁻¹. Concentrations of dissolved inorganic carbon and N₂ were close to atmospheric equilibrium in uncontaminated groundwater, but were more than 2 times higher within the contaminant plume. Excess CO₂ and N₂ suggested in situ formation with a stoichiometry of C and N mineralized via denitrification of 0.8 (C/N). Denitrification within the aquifer resulted in an increase in the natural δ¹⁵N of NO₃⁻ (from +13.6 to +42.0%.) and the N₂ produced, with an isotopic enrichment factor, ϵ, of −13.9%.. Vertical profiles of NH₄⁺ and δ¹⁵N of NH₄⁺ indicated that dissimilatory reduction of NO₃⁻ to NH₄⁺ was also occurring but mass balance calculations indicated that denitrification was the predominant process. These results demonstrate that a combination approach using field mass balance, stable isotope analysis, and laboratory incubations yields useful insight as to the significance of denitrification in aquifer sediments and that closely spaced vertical sampling is necessary to adequately quantify the processes controlling C and N transport and transformation within these environments.