Various in vitro-inhibitors were added with 3H-benzo(a)pyrene (BP) into the perfusion fluids in isolated rat lung perfusions to see whether their effects are dependent on the integrity of tissue. 3H-BP and its metabolites were measured by thin-layer chromatography and radiometry from both samples of perfusion medium and homogenates of lung tissue. The total covalent binding to lung tissue was used as a measure of the formation of reactive metabolites. In methylcholanthrene-induced rat lung, the metabolism of BP was inhibited by alpha-naphthoflavone, an inhibitor of monooxygenase, and less with diethylmaleate, a depletor of glutathione, with salicylamide, an inhibitor of conjugases, and, astonishingly, with D-saccharo-1,4-lactone, an inhibitor of beta-glucuronidase. With trichloropropene oxide, which inhibits epoxide hydratase, the metabolism was either decreased or unchanged. Nicotine had no effect on BP-metabolism. Nicotine and diethylmaleate increased statistically significantly and alpha-naphthoflavone and salicylamide decreased the covalent binding of radioactivity to lung tissue. In most cases, the changes in BP metabolism observed during perfusion can be explained on the basis of effects of modifiers on the enzyme systems.