Endogenous production of N-nitrosoproline (NPRO) was demonstrated in human subjects ingesting a diet low in nitrate and NPRO. The daily endogenous synthesis of NPRO was 26 +/- 10 nmol/day (mean +/- SD). Upon administration of nitrate and proline, the NPRO excreted in urine ranged from 50-318 nmol/24 h. It was found that ascorbic acid and alpha-tocopherol did not lower the background endogenous synthesis of NPRO; however, ascorbic acid was very effective in preventing the nitrate-induced synthesis of NPRO in all subjects, while alpha-tocopherol was less effective in some subjects. Furthermore, it was demonstrated that administered 15N-nitrate could be incorporated into NPRO, suggesting clearly that nitrosation reactions indeed occur in humans. Ascorbic acid significantly inhibited the incorporation of 15N-nitrate into NPRO. The presence of another N-nitrosamino acid, N-nitrosothiazolidine-4-carboxylic acid (NTCA), was detected in the urine of many subjects even without nitrate intake. However, upon ingestion of nitrate, there was a six-fold increase in mean NTCA synthesis (25 +/- 16 nmol/24 h), and all subjects had detectable levels of NTCA in the urine. Ascorbic acid completely blocked the nitrate-induced synthesis of NTCA, while alpha-tocopherol was not as effective.