Previous studies have shown that an aqueous smokeless tobacco extract when administered in a single oral dose to rats results in an enhanced induction of hepatic lipid peroxidation, hepatic DNA single strand breaks, and a marked increase in the urinary excretion of the lipid metabolites malondialdehyde, formaldehyde, acetaldehyde, and acetone. These observations strongly suggest that STE induces the production of reactive oxygen species. We have therefore examined the effects of STE in vivo in rats on the production of nitric oxide (NO) by isolated peritoneal exudate (macrophage) cells and when incubated with cultured J774A.1 macrophage cells. In both cases, a significant increase in NO production was observed. When the antioxidant vitamin E succinate was preadministered to rats, a marked decrease in NO production in response to STE by isolated peritoneal macrophages was observed. Similar results were observed when J774A.1 macrophages were cultured in the presence of vitamin E succinate and STE. When vitamin E succinate alone was cultured with macrophages, an increase in NO production was observed. A similar increase was observed when the vitamin E succinate was administered to rats, and NO production by isolated peritoneal macrophages was assessed. The results demonstrated that the increase in NO production by macrophages in response to vitamin E succinate was due to a succinate moiety. Taken together with previous studies, the results indicate that STE activates macrophages, which result in the production of reactive oxygen species. These reactive oxygen species may be responsible for tissue damaging effects including lipid peroxidation and DNA damage, which may be associated with the cytotoxicity and mutagenicity of smokeless tobacco products.