Disulfiram (DS) has been clinically used to treat recovering alcoholics for over 50 years. It exerts its pharmacologic effect by inhibiting hepatic aldehyde dehydrogenase (ALDH). It has been proposed that DS inhibits ALDH by forming an intermolecular mixed disulfide, presumably with the active site thiols. It has also been shown that reactive sulfur species are produced during DS and H2S metabolism. Here, we have shown that H2S, at or above 50 µM inhibits yeast ALDH, in an in vitro system. There was a dose-response relationship for this effect as increasing H2S concentration further decreased ALDH activity. The decrease in ALDH activity is enhanced if H2S is incubated with ALDH for 30 minutes. Cytochrome C, a hemoprotein and/or some metal cations increased ALDH inactivation by H2S. H2S is, therefore, a weak inhibitor of ALDH and requires a catalyst to convert it into a more active species. It is postulated that heme or metal cations catalyse the oxidation of H2S to reactive sulfur species which can form persulfides with the ALDH cystein at the catalytic site and inhibit its activity. ALDH activity was restored by adding reducing agents such as dithiothreitol and glutathione to reduce the cysteine persulfide moiety of the enzyme. H2S also increased the cytotoxicity of acetaldehyde and/or 2-chloroacetaldehyde in the presence of the alcohol dehydrogenase inhibitor 4-methylpyrazole towards freshly isolated rat hepatocytes. ALDH inhibition by H2S indicates that the H2S must have an “antabuse” effect which would suggest unpleasant reactions to alcohol in the people exposed to H2S gas.