Ethacrynic acid, a potent inhibitor of glutathione S-transferases (GST), has been shown to enhance the cytotoxicity of chlorambucil in drug resistant cell lines, but a definite mechanism has not been established. Both covalent binding to GST and reversible inhibition of GST have been reported. In the present study no irreversible inhibition was observed: for all rat GST tested, inactivation was complete within 15 sec at 0°, and dialysis of GST after incubation with ethacrynic acid gave complete recovery of enzyme activity for all isoenzymes tested. Moreover, the inhibition was competitive towards 1-chloro-2,4-dinitrobenzene and non-competitive towards glutathione for rat isoenzyme 1-1. Strong inhibition of both human and rat GST of the α-, μ- and π-classes was obtained with ethacrynic acid, while conjugation of ethacrynic acid with glutathione did not abolish its inhibiting properties. For the α-, μ- and π-class i₅₀ values (μM) were 4.6–6.0, 0.3–1.9 and 3.3–4.8, respectively for ethacrynic acid, and 0.8–2.8, < 0.1–1.2 and 11.0, respectively for its glutathione conjugate. Of all isoenzymes tested the human isoenzyme μ is most sensitive to the action of both ethacrynic acid and its glutathione conjugate.