Aquifer denitrification is among the most poorly constrained fluxes in global and regional nitrogen budgets. The few direct measurements of denitrification in groundwaters provide limited information about its spatial and temporal variability, particularly at the scale of whole aquifers. Uncertainty in estimates of denitrification may also lead to underestimates of its effect on isotopic signatures of inorganic N, and thereby confound the inference of N source from these data. In this study, our objectives are to quantify the magnitude and variability of denitrification in the Upper Floridan Aquifer (UFA) and evaluate its effect on N isotopic signatures at the regional scale. Using dual noble gas tracers (Ne, Ar) to generate physical predictions of N2 gas concentrations for 112 observations from 61 UFA springs, we show that excess (i.e. denitrification-derived) N2 is highly variable in space and inversely correlated with dissolved oxygen (O2). Negative relationship between O2 and ??15NNO 3 across a larger dataset of 113 springs, well-constrained isotopic fractionation coefficients, and strong 15N: 18O covariation further support inferences of denitrification in this uniquely organic-matter-poor system. Despite relatively low average rates, denitrification accounted for 32% of estimated aquifer N inputs across all sampled UFA springs. Back-calculations of source ??15NNO 3 based on denitrification progression suggest that isotopically-enriched nitrate (NO3-) in many springs of the UFA reflects groundwater denitrification rather than urban- or animal-derived inputs. ?? Author(s) 2011.