The nature of the reactive metabolite of 3-methylindole is investigated using microsomal preparations prepared from the lungs of cattle. Nucleophilic thiol agents, glutathione, L-cysteine and N-acetyl-L-cysteine, protected microsomal proteins against alkylation by the reactive metabolite of 3-methylindole. The cytosol fraction from the lungs of cattle increased the protective effect of these thiol agents. Pretreatment of sheep with diethylmaleate, which depletes glutathione, increased the severity of the pneumotoxic effect of 3-methylindole, whereas pretreatment with L-cysteine decreased the severity of this effect. These findings are consistent with a hypothesis that an electrophilic reactive metabolite of 3-methylindole is responsible for its pneumotoxic effect and implies that glutathione and glutathione S-transferases are involved in the detoxification of this reactive metabolite. Nucleophilic thiol agents can be useful in the prevention of reactive metabolite induced-lung injury.