Repetitive oral propranolol administration to rats (100 mg/kg/day for 5 days) resulted in an 80% inhibition of propranolol Type I spectral binding capacity. This paralleled a similar reduction in the microsomal metabolism of propranolol when incubated at low substrate concentrations (less than 2 microM). Propranolo was converted both in vitro and in vivo by a microsomal mixed function oxidase to a reactive intermediate metabolite capable of covalently binding with microsomal macromolecules. We propose that covalent binding of the intermediate to the catalytic site of a form(s) of cytochrome P-450 that metabolizes propranolol would account for the marked inhibition of propranolol metabolism observed following propranolol pretreatment.