Although tobacco chewing is strongly associated with a high risk of oral and upper alimentary tract cancers, the nature of mutagenic exposure among users has not been clearly defined. In this study, tobacco-specific and mutagenic exposure of chewers of tobacco with lime was evaluated by analysis of gastric fluid (GF). The pH, nitrite and cotinine levels of GF samples from chewers and non-chewers were determined and the samples were tested for mutagenicity in the Ames Salmonella/microsome assay using Salmonella typhimurium strains TA98, TA100 and TA102. Cotinine was not detected in GF from non-chewers while the levels ranged between 0.4-13.64 micrograms/ml in samples from chewers; however, the mean pH values (3.8 +/- 0.4 versus 2.8 +/- 0.3) and nitrite levels (29.40 +/- 1.51 versus 27.39 +/- 0.83 microM) were similar in both groups. While all GF samples from non-chewers were non-mutagenic, samples from chewers were directly mutagenic or upon nitrosation to all the three tester strains and to TA102 strain in the presence of S9. Experiments using scavengers of reactive oxygen species (ROS) showed that mannitol and benzoate abolished the mutagenic response of TA102, indicating that ROS are principally responsible for oxidative damage. The findings provide specific information regarding the mutagenic exposure among tobacco chewers and suggest that tobacco chewing may be an important risk factor in the development of gastric cancer.