A novel enzyme catalyzing cleavage of the thioether linkage in cysteine conjugates of aromatic compounds, such as 2,4-dinitrobenzene and p-bromobenzene, has been purified about 500-fold from rat liver cytosol. Incubation of S-(2,4-dinitrophenyl)cysteine with the enzyme preparation yielded 2,4-dinitrobenzenethiol, pyruvic acid, and NH3 at equimolar ratios, indicating that the thioether cleavage probably proceeds via an alpha,beta elimination reaction. The thiol product was methylated and the methylated derivative 1-methylthio-2,4-dinitrobenzene, was identified by mass spectrometry and proton NMR spectroscopy. The Km value of S-(2,4-dinitrophenyl)cysteine was 0.5 mM at pH 7.4 in phosphate buffer. The enzyme activity was inhibited by hydroxylamine. No cofactor requirement was observed. A combination of the partially purified enzyme and hepatic microsomes that contain thiol methyltransferase (EC 2.1.1.9) converted cysteine conjugates of 2,4-dinitrobenzene and p-bromobenzene to the corresponding methylthio-containing metabolites; S-adenosylmethionine was required. An important role of this novel beta-lyase enzyme in the formation of methylthio-containing metabolites of various drugs is indicated.