1. 2-(Allylthio)pyrazine (2-AP) has been demonstrated to protect the liver against toxicants by inhibiting CYP2E1 activity. Since 2-mercaptopyrazine (2-MP) is presumed to be a metabolite of 2-AP, the experiments were performed to determine whether rat liver microsomal and/or cytosolic preparations could catalyse the S-methylation of 2-MP. 2. It was found that both rat liver microsomes and cytosol could catalyse the S-methylation of 2-MP. The microsomal activity displayed biphasic substrate kinetics, with apparent Km = 8.44+/-2.68 and 417+/-74 microM for the high- and low-affinity activities respectively. The high-affinity activity had an apparent Km for S-adenosyl-L-methionine (Ado-Met) of 3.52 microM. The cytosolic activity also displayed biphasic substrate kinetics, with apparent Km of 3.26+/-0.62 and 91.6+/-23.1 microM for the high- and low-affinity activities respectively. 3. The microsomal S-methylation of 2-MP was inhibited by 2,3-dichloro-alpha-methylbenzylamine (DCMB), SKF-525A and benzylamine, known microsomal thiol methyltransferase (TMT) inhibitors, whereas cytosolic activity was inhibited by anisic acid and 3-chlorobenzoate, which also inhibit cytosolic thiopurine methyltransferase (TPMT). Both activities were inhibited by S-adenosyl-L-homocysteine (Met-Hcy). 4. These results suggest that both TMT and TPMT may be involved in the in vivo methylation of 2-MP.