The mechanisms regulating NO-3 concentration and dissimilation in a shallow, spring-fed well were investigated by creating variable flow conditions in a series of sediment-filled laboratory microcosms. Nitrate concentrations in the laboratory microcosms reproduced field observations; effluent concentrations were as high as 100% of the influent concentration when flow rates were high, less than 10% of the influent concentration when flow rates were low, and of intermediate concentration when flow rates were intermediate. The NH+4 concentration (up to 6.9 mg NH+4-N L-1) did not vary despite changes in flow, which indicated that assimilatory and dissimilatory NO-3 reduction to NH+4 were inconsequential. Changes in NO-3 concentration were consistent with dissimilatory reduction of NO-3 to N2 gas (denitrification) based on the following evidence: intermediates of denitrification accumulated with short NO-3 retention times (flow rates > 2 mL min-1) and disappeared with long NO-3 retention times (flow rates < 0.4 mL min-1); between 60 and 68% of the influent NO-3 was unaccounted for at the end of incubation, presumably because it was denitrified; decreasing sediment content in the microcosms (from >1000 g to <400 g) to shorten retention time decreased NO-3 reduction; sediment pH increased from pH 7.5 to 8.5, which would occur during denitrification, during periods of low flow; acetylene caused N2O to accumulate, indicating that the terminal step in denitrification was blocked. Forty-seven percent of the springs, wells, and tiles from a statewide water assessment in Kentucky demonstrated variable NO-3 concentration. Denitrification appears to affect water quality assessment of NO-3 concentration during the year and attenuate NO-3 impacts on shallow groundwater.