This study was designed to investigate the metabolism of methanethiol, and the involvement of methanethiol and its metabolites in the transamination pathway of methionine. Gaseous methanethiol, methanethiol-mixed disulfides and dimethylsulfide were formed from 3-methylthiopropionate, a metabolite in the transamination pathway of methionine, during incubation with human and rat hepatocytes. An increase of the 3-methylthiopropionate concentration resulted in an increased formation of the products, up to a substrate concentration of 4.4 mM. Higher substrate levels resulted in a decreased methanethiol formation, probably due to poisoning of the system. However, in human hepatocytes the formation of dimethylsulfide increased up to a 3-methylthiopropionate concentration of 12.5 mM. The formation of methanethiol, dimethylsulfide and methanethiol-mixed disulfides from 3-methylthiopropionate in hepatocytes of both human and rat support the hypothesis that methanethiol can be formed from methionine via the transamination pathway.