The potential for denitrification was tested in water samples from four Upper Floridan aquifer wells near a reclaimed water application site, in west Orange County Florida, and two adjacent springs. Results of the study indicate that denitrifying bacteria are present in the groundwater and spring water samples, and that these bacteria can readily denitrify the waters when suitable geochemical conditions exist. The acetylene block technique was used to assess nitrous oxide in the samples that was produced by denitrification. The laboratory incubation experiment consisted of four different treatments to each of the six samples: (1) ambient water (no added nitrate or glucose), (2) ambient water amended with 1.4 milligrams per liter (mg/L) nitrate as nitrogen (N), (3) ambient water amended with 5.0 mg/L nitrate as N, and (4) ambient water amended with 5.0 mg/L nitrate as N and 10 mg/L glucose as C6H12O6. A companion set of incubations using treatment 2 tracked changes in nitrate and nitrite concentration with time. The rate of denitrification in treatment 2 ranged from 0.059 to 0.124 milligram per liter per day nitrogen [(mg/L)/d N] and in treatment 3 ranged from 0.071 to 0.226 (mg/L)/d N. At all of the sampling sites, treatment 4 yielded denitrification rates at least an order of magnitude greater than those measured for the other treatments; rates ranged from 2.3 to 4.4 (mg/L)/d N. The electron donor supply, dissolved organic carbon, in the groundwater and springwater is sufficient to remove at least 1.1-1.4 mg/L nitrate as N in 20 to 30 days, as indicated by nitrous oxide production rates under ambient conditions (treatment 1). The even higher nitrate removal observed with addition of supplemental carbon in treatment 4 suggests that carbon is a limiting nutrient in this reaction. Denitrifying activity might explain the low ambient nitrate concentrations in the Upper Floridan aquifer in this area.