The kinetics and reversiblity of the suicide inactivation of rat liver cytochrome P-450 by chloramphenicol [an antibiotic] were investigated with the use of a reconstituted monooxygenase system purified from liver microsomes of phenobarbital-treated rats. At a ratio of 1 U of NADPH-cytochrome P-450 reductase/nm of cytochrome P-450 and a chloramphenicol concentration of 1 mM, the t1/2 [half-life] for the inactivation of cytochrome P-450 is < 2 min. The inactivated cytochrome regains some of its activity upon incubation at 25.degree. or 37.degree., and experiments with [14C]chloramphenicol show that this partial reactivation is accompanied by the release of some of the 14C originally bound covalently to the cytochrome P-450. The 14C-labeled material spontaneously released from 14C-labeled cytochrome P-450 is in the form of oxalic acid, and the latter is derived from a hydroxylamine-labile adduct of chloramphenicol and cytochrome P-450. The 14C-labeled material released by hydroxylamine was identified as the hydroxamic acid of oxalic acid. Trapping experiments with cysteine suggest that the adduct, the spontaneous degradation of which appears to be involved in the reactivation of cytochrome P-450, contains an ester rather than a thioester linkage between cytochrome P-450 and a metabolite of chloramphenicol. This metabolite may not be identical with chloramphenicol oxamyl chloride, which was the active metabolite implicated in the formation of the 50% covalently bound material which was stable to hydroxylamine treatment.