Denitrification and nitrogen transport were quantified in a sandy glacial aquifer receiving wastewater from a septage-treatment facility on Cape Cod, MA. The resulting groundwater plume contained high concentrations of NO3- (32 mg of NL-1), total dissolved nitrogen (40.5 mg of N L-1), and dissolved organic carbon (1.9 mg of C L-1) and developed a central anoxic zone after 17 months of effluent discharge. Denitrifying activity was measured using four approaches throughout the major biogeochemical zones of the plume. Three approaches that maintained the structure of aquifer materials yielded comparable rates: acetylene block in intact sediment cores, 9.6 ng of N cm-3 d-1 (n = 61); in situ N2 production, 3.0 ng of N cm-3 d-1 (n = 11); and in situ NO3- depletion, 7.1 ng of N cm-3 d-1 (n = 3). In contrast, the mixing of aquifer materials using a standard slurry method yielded rates that were more than 15-fold higher (150 ng of N cm-3 d-1, n = 16) than other methods. Concentrations and ??15N of groundwater and effluent N2, NO3-, and NH4+ were consistent with the lower rates of denitrification determined by the intact-core or in situ methods. These methods and a plumewide survey of excess N2 indicate that 2-9% of the total mass of fixed nitrogen recharged to the anoxic zone of the plume was denitrified during the 34-month study period. Denitrification was limited by organic carbon (not NO3-) concentrations, as evidenced by a nitrate and carbon addition experiment, the correlation of denitrifying activity with in situ concentrations of dissolved organic carbon, and the assessments of available organic carbon in plume sediments. Carbon limitation is consistent with the observed conservative transport of 85-96% of the nitrate in the anoxic zone. Although denitrifying activity removed a significant amount (46250 kg) of fixed nitrogen during transport, the effects of aquifer denitrification on the nitrogen load to receiving ecosystems are likely to be small (<10%).