2,3,5-(Triglutathion-S-yl)hydroquinone [2,3,5-(triGSyl)HQ] is a potent nephrotoxicant when administered to male rats. We now report that significant species differences exist in susceptibility to 2,3,5-(triGSyl)HQ-mediated nephrotoxicity. Metabolism of glutathione conjugates involves cleavage of teh glutamate and glycine moieties by gamma-glutamyltranspeptidase (gamma-GT) and dipeptidases, respectively, and the nephrotoxicity of 2,3,5-(triGSyl)HQ can be prevented by the inhibition of renal gamma-GT. The resulting cysteine conjugate exhibits a balance between N-acetylation, and N-deacetylation of the mercapturic acid biosynthesis in various species contribute to species susceptibility to 2,3,5-(triGSyl)HQ. Renal gamma-GT activity toward 2,3,5-(triGSyl)HQ was highest in the rat (Fischer 344 and Sprague-Dawley) and consistent with the sensitivity of this species to 2,3,5-(triGSyl)HQ (20 micromol/kg iv)-mediated nephrotoxicity. The gamma-GT-mediated hydrolysis of 2,3,5-(triGSyl)HQ was similar in B6C3F1 and BALB/c mice and guinea pigs. In these species, the gamma-GT activity ranged between 30-45% of the activity measured in rats. Although, the activity of gamma-GT was similar in mice and guinea pigs, only guinea pigs were susceptible to 2,3,5-(triGSyl)HQ (200 micromol/kg iv)-induced renal necrosis. The gamma-GT-mediated hydrolysis of 2,3,5-(triGSyl)HQ was lowest in the hamster, and this species were not susceptible to the renal toxicity of this conjugate. Thus, factors in addition to gamma-GT activity probably contribute to species susceptibility to 2,3,5-(triGSyl)HQ nephrotoxicity. The kinetics of the AT-125-mediated inhibition of gamma-GT differed between species, indicative of potential differences in the regulation of gamma-GT. Consistent with this view, the ratio between the hydrolysis and transpeptidation of 2,3,5-(triGSyl)HQ varied 10-fold between the species examined, and was highest in the guinea pig (0.48) and lowest in the hamster (0.05). Guinea pigs also exhibited the highest renal cytosolic N-deacetylase activity and the lowest N-acetylase activity. The ratios of N-deacetylation to N-acetylation in guinea pigs, BALB/c mice, B6C3F1 mice, hamsters, Fischer 344 rats, and Sprague-Dawley rats were 4.57, 0.16, 0.14, 0.04, 0.03, and 0.02, respectively. Because quinol-cysteine conjugates seem to undergo oxidation more readily than the corresponding mercapturates, the balance of N-deacetylase and N-acetylase in the guinea pig may contribute to the susceptibility of this species to 2,3,5-(triGSyl)HQ nephrotoxicity.