After incubation of [ 14C]imipramine with rat liver microsomcs up to 0–7 mole/mg was irreversibly bound per mg of microsomal protein. If albumin was added to the microsomal incubations [ 14C]imiprámine was also irreversibly bound to this protein. The irreversible binding of imipramine to protein was determined by exhaustive solvent extraction and charcoal adsorption, and measurement of the remaining 14C-radioactivity in the protein. The binding reaction was dependent on oxygen, NADPH, microsomal protein content and substrate concentration. It was inhibited by CO and SKF 525-A. Pretreatment of rats with phenobarbital did not increase the amount of imipramine irreversibly bound to protein. Glutathione and other cysteine derivatives diminished the binding, whereas incubation with the epoxide hydrase inhibitor trichloropropene oxide resulted in an increase of imipramine irreversibly bound to protein. The results favour the concept that irreversible protein binding of imipramine is catalyzed by a cytochrome P-450-dependent hydroxylation via an epoxidation step. Irreversible protein binding of imipramine was also detectable with three samples of human liver microsomes.