Marked interspecies variability exists in the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), with the rat having an LD50 about 25-fold greater than the guinea pig. The metabolism of TCDD was examined by incubating hepatocytes isolated from these animals with purified [14C]TCDD (2.2 microM) for 8 hr. Over the 8-hr incubation, cytochrome P-450 content and ethoxyresorufin O-deethylase and benzphetamine N-demethylase activities were well maintained, indicating the functional viability of the hepatocytes. Quantitative differences were observed in the rate of [14C]TCDD metabolism, with hepatocytes from control rats metabolizing TCDD at a rate 2.8-fold greater than hepatocytes from control guinea pigs. The role of the hepatic cytochrome P-450-448-dependent monooxygenase system in the metabolism of TCDD was examined through the use of hepatocytes isolated from animals pretreated with either TCDD (5 micrograms/kg, ip; 72 hr prior to hepatocyte isolation) or phenobarbital (80 mg/kg, ip X 3 days; 24 hr prior to isolation). The rate of [14C]TCDD metabolite formation in hepatocytes from TCDD pretreated guinea pigs (0.26 +/- 0.14 pmol mg cell protein-1 hr-1) was unchanged from the control rate (0.25 +/- 0.07), while the rate in hepatocytes from TCDD pretreated rats (2.26 +/- 0.43 pmol mg-1 hr-1) was 3.2-fold greater than control (0.70 +/- 0.10) and nine times greater than in hepatocytes from TCDD-pretreated guinea pigs. In addition, significant differences were observed in the profiles of the metabolites formed by hepatocytes from TCDD-pretreated rats and guinea pigs. On the other hand, phenobarbital pretreatment produced little change in the rate of [14C]TCDD metabolism in rat hepatocytes (0.98 +/- 0.13 pmol mg-1 hr-1). These results suggest that TCDD may be metabolized by a TCDD inducible form of cytochrome P-448 which is expressed in the rat but not in the guinea pig. Furthermore, the differences in the hepatic metabolism of TCDD in the rat and guinea pig and in the ability of TCDD to induce its own rate of metabolism may play a major role in explaining the varying susceptibility of these species to the acute toxicity of TCDD.