Cholangiocarcinoma (CCA) is one of the most prevalent cancers in north-east Thailand and has been associated with infestation by the liver fluke Opisthorchis viverrini (OV). Two samples of 12-h overnight urine (after dosing with 500 mg proline and 200 mg ascorbic acid or 500 mg proline alone) were collected from about 100 inhabitants in five contrasting incidence areas for CCA and hepatocellular carcinoma. The incidences of CCA and hepatocellular carcinoma were not correlated with either the amount of NPRO or other nitrosamino acids, endogenous nitrosation potential (difference in NPRO levels between proline dose and proline and ascorbic acid dose), or nitrate level. However, when urinary levels of nitrosamino acids were compared in subjects living in high-risk areas, subjects who were positive for OV antibody excreted significantly more (p less than 0.01) NPRO (12.3 +/- 18.7 micrograms/12 h) after proline ingestion than those who were negative 3.5 +/- 3.2 micrograms/12 h). After ingestion of ascorbic acid, the NPRO levels in the positive subjects were significantly reduced (p less than 0.01) to 2.4 +/- 2.0 micrograms/12 h, suggesting that endogenous nitrosation of proline was inhibited. Thus, endogenous nitrosation potential estimated from the difference of NPRO and sum of nitrosamino acids excreted in the two urine samples was significantly higher in subjects positive for the OV antibody. In addition, of the representative food samples and beverages consumed frequently in high-risk areas for CCA, fermented fish and pork contained N-nitrosodimethylamine (0-26 micrograms/kg), N-nitrosopyrrolidine (0-117 micrograms/kg) and N-nitrosopiperidine (0-23 micrograms/kg).(ABSTRACT TRUNCATED AT 250 WORDS)