We have demonstrated that ammonia is oxidized to nitrate in the rat. Male Sprague-Dawley rats gavaged with 1 000 mumol 15N-ammonium chloride each day for five days were found to excrete low, but significant, amounts of excess 15N-nitrate in their urines on the five days of treatment and on the five subsequent days. We recovered a total of 0.28 +/- 0.03 mumol excess 15N-nitrate (mean +/- SE) per rat, which indicates that ammonia is converted to nitrate with a yield of about 0.0080%. 15N-Hydroxylamine was oxidized in the rat to 15N-nitrate with a yield of 4.7%, but oxidation of 15N-labeled glycine and L-glutamic acid to 15N-nitrate could not be detected. These results suggest that hydroxylamine, but not glycine or L-glutamic acid, may be an intermediate in the ammonia oxidation process. The injection of rats with Arochlor 1254 failed to stimulate nitrate synthesis, which indicates that the cytochrome P-450 drug metabolizing system is probably not involved in ammonia oxidation. Carbon tetrachloride, which causes hepatic lipid peroxidation, produced a small, but significant, increase in nitrate synthesis. Our results are consistent with the hypothesis that ammonia is oxidized in vivo by a non-enzymatic process involving reactive oxygen species. We estimate that a 215 g rat produces 3.0 mumol of nitrate per day by this process. The significance of our results to the problem of endogenous N-nitroso compound formation in man is discussed.