Repetitive oral administration of propranolol to rats (100 mg/kg/day for 5 days) resulted in a marked inhibition of hepatic microsomal metabolism of propranolol when incubated at low initial substrate concentrations (less than 2 microM). Associated with the inhibition of propranolol metabolism was a significant reduction in metabolites derived from naphthalene ring oxidation and an increased formation of N-desisopropylpropranolol. In vivo studies after propranolol pretreatment resulted in: an increased hepatic concentration and an increased systemic availability of propranolol; a decreased hepatic and plasma concentration of polar metabolites; and an increased plasma concentration of metabolites derived from propranolol N-dealkylation. Propranolol was converted both in vitro and in vivo by a hepatic microsomal mixed-function oxidase to a reactive metabolite capable of covalently binding with microsomal macromolecules. We propose that selective covalent binding of the reactive intermediate to the molecular form of cytochrome P-450 that ring hydroxylates propranolol would account for the marked inhibition of propranolol metabolism in vitro and for the increased systemic availability of propranolol in vivo after pretreatment.