Abstract The irreversible and reversible inhibition of glutathione S-transferases (GSTs) by eugenol was studied in rat, mouse and man. Using liver cytosol of human, rat and mouse, species differences were found in the rate of irreversible inhibition of GSTs by eugenol in the presence of the enzyme tyrosinase. Tyrosinase was used to oxidize eugenol. No inhibition was observed in the absence of tyrosinase. The rate of irreversible inhibition of GSTs was highest in mouse cytosol, and lowest in rat cytosol. In addition, the irreversible inhibition of human and rat GSTs by eugenol was studied using purified isoenzymes of man and rat. The human GST isoenzymes A1-1, M1a-1a and P1-1 and the rat GST isoenzymes 1-1, 2-2, 3-3, 4-4 and 7-7 were irreversibly inhibited by eugenol in the presence of tyrosinase. In this respect human GST PI-1 and rat GST 7-7 were by far the most sensitive enzymes; human GST A2-2 was not inhibited. Indications were found that human GST P1-1 may be inhibited via three mechanisms: in addition to the well documentated nucleophilic addition of quinones and oxidation of cysteine residues, a covalent subunit cross-linking was also observed. The reversible inhibition of human and rat GST by eugenol, eugenol methyl ether, isoeugenol methyl ether, 2-allylphenol and 4-propylphenol was also studied using purified isoenzymes. The reversible inhibition of human and rat GSTs, using 1-chloro-2,4-dinitrobenzene as substrate, was expressed as I 25. All compounds caused moderate reversible inhibition (I 25 ranged from 0.2 to 5.4 mM for human GSTs and from 0.4 to 4.9 mM for rat GSTs). In rat, eugenol methyl ether was the strongest inhibitor. In human, the overall inhibiting capacities of eugenol, eugenol methyl ether, isoeugenol methyl ether and 4-propyl phenol were more or less similar; 2-allylphenol was the poorest inhibitor.