The drug disulfiram (DSF, Antabuse®) has been used in the therapy of alcohol abuse. It is a potent inhibitor of aldehyde dehydrogenase. Its reduced form, diethyldithiocarbamate (DDTC), and further metabolites show similar activities. DSF and DDTC have also been widely used to inhibit mixed-function oxidases. In this study, the reversible inhibition and time-dependent inactivation of the major rat and human glutathione S-transferase (GST) isoenzymes by DSF and DDTC was investigated. Reversible inhibition, using 1-chloro-2,4-dinitrobenzene as substrate for the GST alpha-, mu-, and pi-class, expressed as I50 (in μM), ranged from 5-18 (human A1-1), 43-57 (rat 4-4) and 66-83 (rat 1-1), for both DSF and DDTC. The I50 for rat GST theta, using 1,2-epoxy-3-(p-nitrophenoxy)-propane as substrate, was 350 μM for DDTC. The other GSTs were significantly less sensitive to inhibition. The major part of reversible inhibition by DSF was shown to be due to DDTC, formed rapidly upon reduction of DSF by the glutathione (GSH) present in the assay to measure GST activity. The oxidized GSH formed upon reduction of DSF might also have made a minor contribution to reversible inhibition. The rat and human pi-class was, by far, the most sensitive class for time-dependent inactivation by DSF, but no such inactivation was observed for any of the GSTs by DDTC. Moderate susceptibility to inactivation by DSF of all the other GSTs was observed, except for human A2-2, which does not possess a cysteine residue. Consistent with the assumption that a thiol residue is involved in this inactivation, a significant part of the activity could be restored by treatment of the inactivated GST with GSH or dithiotreitol.