Two kinds of enzymes (tentatively designated methyl sulfoxide reductases I and II) responsible for the reduction of the methyl sulfoxide group on various xenobiotics have been purified about 223- and 155-fold, respectively, from rat kidney cytosol. The molecular weight was determined to be 12,000 +/- 1000 for methyl sulfoxide reductase I and 24,000 +/- 1000 for methyl sulfoxide reductase II. Thioredoxin or dithiothreitol is essential in order for the reducing activity to occur. The respective Km values of p-bromophenylmethyl sulfoxide were 2.75 and 1.30 mM for methyl sulfoxide reductases I and II. Replacement of the methyl group on the sulfur atom with a longer alkyl group or phenyl group caused a markedly low or negligible substrate activity.