3-Methylindole (3MI) selectively causes damage to pulmonary tissues; the species-selective order is goats, rats, and rabbits, with rabbits sustaining the least damage. 3MI is bioactivated to toxic intermediates by cytochrome P450 enzymes. Covalent binding of the electrophilic 3-methyleneindolenine intermediate to proteins is a likely mechanism of 3MI-mediated lung damage. Polyclonal antibodies were developed to thioether adducts of 3-methyleneindolenine and were shown by competitive enzyme-linked immunosorbent assay (ELISA) to be highly selective for the detection of 3MI adducts. Rabbits, rats, and goats were treated with 350, 400, and 15 mg/kg 3MI, respectively. The lungs, liver, and kidneys of each animal were collected 24 hr later and tissue fractions were analyzed by ELISA. Lung tissue fractions from goat (pellet, cytosol, and microsomes) had greater immunoreactivity than those from rat. Immunoreactivity in rat tissues was greater than that in rabbit tissues. In all of the animals, lung had greater immunoreactivity than kidney, and kidney had greater reactivity than liver. These studies demonstrate that thioether adducts of 3MI with proteins can be detected specifically by these antisera, and the adducts are precisely correlated to species and tissue susceptibility of 3MI. In addition, human lung and liver samples were moderately immunoreactive. Therefore, humans form adducts of 3MI in these tissues and are predicted to be susceptible to 3MI-mediated toxicity.