Epidermoid cancers of tracheal origin produced in Syrian golden hamsters and Fischer strain 344 rats are models for human bronchogenic carcinomas. These two species differ, however, in the sensitivity of their tracheal epithelia to tumor induction elicited by intratracheal benzo[a]pyrene (BP)-ferric oxide administration. The tracheas of hamsters are quite sensitive to the carcinogenic effects of BP-ferric oxide, but rat tracheas are apparently resistant to effects of comparable treatments by this route of administration. Rat tracheas are not completely resistant to polynuclear hydrocarbon carcinogenesis because in the heterotopic tracheal graft model, epidermoid carcinomas have been produced frequently. To determine whether differences in BP metabolism could explain this difference between species, quantitative kinetic and chromatographic studies of benzo[a]pyrene monoxygenase activity were carried out in epithelial microsomes and cells from organ cultures of rat and hamster tracheas. The Vmax was 2-fold greater in hamster tracheal cells than in rat tracheal cells whereas the Km values were identical. H.p.l.c. profiles from microsomes of rat and hamster tracheal epithelial cells incubated with BP exhibited extreme differences. Hamster tracheal microsomes produced large proportions of BP-quinones, BP-phenols, and BP-diols but rat tracheal microsomes produced mostly 3-OH BP. The total metabolic rate for BP in rat tracheal organ cultures was half that in cultures of hamster tracheas. The metabolites isolated in organ cultures of hamster and rat tracheas well reflected secondary reactions of conjugation and recycling. When evaluated with respect to the amount of tissue used, the most striking difference between rat and hamster tracheal organ cultures was in the amount of products which co-chromatographed with bay-region BP-tetrols. The amount of BP-tetrols produced by hamster tracheas was 0.22 pmol/mg tissue/24 h, and by rat, 0.012 pmol/mg tissue/24 h. The level of BP-DNA binding (in pmol/microgram DNA/24 h) catalyzed by hamster tracheal cells was 26.6 +/- 11.4, and by rat tracheas was 1.55 +/- 1.29. These interspecies differences associated with the formation of BP-diol epoxide, a presumed ultimate carcinogenic form of BP, are consistent with the differences between these two rodent species in susceptibility to carcinogenesis in this tissue.