2-(Methylthio)ethanesulfonate (CH3S-CoM) is formed as an intermediate in methanogenesis from methanol by cell-free extracts of Methanosarcina barkeri. The enzyme system involved in the methyl transfer from methanol to 2-mercaptoethanesulfonate (HS-CoM) was resolved into two enzyme fractions. One enzyme (methanol:5-hydroxy-benzimidazolylcobamide methyltransferase) appears to be a cobalamin-containing protein, which is oxygen sensitive. The other enzyme (Co-methyl-5-hydroxybenzimidazolylcobamide: HS-CoM methyltransferase) was purified. It is insensitive to oxygen and it transfers also the methylgroup from Co-methyl-5,6-dimethylbenzimidazolylcobamide to HS-CoM.