Previous laboratory studies have demonstrated that hippuric acid, a ruminant urine constituent, can mitigate nitrous oxide (N2O) emissions from simulated urine patches. Hippuric acid has the potential to be a N2O mitigation tool because animal diets can be manipulated to adjust its concentration in the urine. This study was conducted to determine if the effect observed in previous laboratory studies would also occur in situ under field conditions. In our field study, plots were treated with unadulterated bovine urine (56 mM hippuric acid), the same bovine urine amended with either benzoic acid (34 mM), dicyandiamide (DCD) or varying rates of hippuric acid (up to 90 mM). Soil inorganic-N, N2O fluxes, and plant N responses were monitored over a 78 d period. Effects on microbial communities were monitored by determining the size and structure of nitrite oxidizer (nxrA) and nitrite reducer (nirS) bacterial populations using real-time PCR and denaturing gradient gel electrophoresis (DGGE), respectively. Decreases in N2O emissions, with increasing hippuric and benzoic acid concentrations, were only seen on Day two of the trial. With the exception of the DCD treatment (0.60% of N applied) the amended urine treatments did not significantly affect emissions of N2O as a percentage of N applied (1.28-1.65%). Soil inorganic-N and plant response were not affected by urinary amendment, except in the DCD treatment where nitrification inhibition occurred. Nitrite oxidizer community structures shifted and increased approximately 5.4-fold in size over 48 d in response to urine, although no specific response to elevated hippuric acid or benzoic acid was observed. No treatment effects were observed on community structure of the nitrite reducing bacteria but averaged over time the highest rate of hippuric acid significantly decreased nirS gene copy numbers g(-1) soil. We concluded that under the conditions of this field study, increasing hippuric or benzoic acid concentrations in bovine urine had no effect on N2O mitigation in situ. We argue that the discrepancy with previous laboratory studies may be related to differences in soil pH, microbial communities and the presence of vegetation. Further research is needed to determine the potential for hippuric acid as a tool to mitigate N2O emissions, and its effect(s) on resident N cycling microorganisms. (C) 2009 Elsevier Ltd. All rights reserved.