3-Methylindole (3MI) is a pneumotoxin that causes selective lung lesions indicative of Clara cell and alveolar epithelial cell damage in ruminants and rodents. The present study examined the cytotoxicity of 3MI to isolated rabbit Clara cells, type II alveolar epithelial cells, and alveolar macrophages. 3MI produced a dose-dependent cytotoxicity to Clara cells detectable within 1 hr of incubation at 37 degrees C which reached a maximum at 3 hr. Concentrations of 0.25 and 0.5 mM 3MI were cytotoxic to Clara cells, while type II and alveolar macrophages required 1 mM 3MI before cytotoxicity was observed. The cytochrome P450 suicide substrate inhibitor, 1-aminobenzotriazole, inhibited 3MI-induced cytotoxicity in Clara cells, type II cells, and alveolar macrophages. These observations were consistent with a cytochrome P450-mediated bioactivation of 3MI to a toxic intermediate. Studies with a trideuteromethyl analog of 3MI demonstrated a much reduced cytotoxicity to Clara cells as well as to type II cells, and macrophages. The deuterium isotope effect suggested that C-H bond breakage at the 3-methyl group is a requisite oxidative transformation in the bioactivation of 3MI to a selective lung cell cytotoxin. The selectivity of cellular cytotoxicity is probably associated with higher rates of bioactivation by Clara cell cytochrome P450 monooxygenases compared to those of type II cells and macrophages. These studies demonstrate that 3MI is bioactivated in isolated pulmonary cells without the intervention of other organs and that bioactivation requires functional cytochrome P450 enzymes.