The effect of phenobarbital treatment on the linkage between carbon tetrachloride-mediated cytochrome P-450 loss and lipid peroxidation in rat liver microsomes was studied. Male Sprague-Dawley rats, pretreated with 3 daily i.p. doses of phenobarbital (50 mg/kg) or saline, were orally dosed with carbon tetrachloride (0.01–2.5 ml/kg), with liver microsomes prepared at 7.5–180 min after carbon tetrachloride treatment. In vivo cytochrome P-450 loss displayed pseudo-first-order kinetics, and the initial rates of diene conjugate formation were saturable with dose. Phenobarbital pretreatment decreased the in vivo t 0.5, max from 27.0 to 15.6 min, and increased the K d, app from 0.78 to 1.30 ml/kg for carbon tetrachloride mediated cytochrome P-450 loss. Phenobarbital had no effect on the in vivo V max (1.03 to 1.04 ΔOD 232 nm/min/mg phospholipid) for carbon tetrachloride mediated diene conjugate formation, but decreased the K m, app from 0.22 to 0.10 ml/kg. These results are consistent with destruction of cytochrome P-450 heme resulting from a metabolite which does not leave the site of generation, and with phenobarbital pretreatment enhancing the initiation of lipid peroxidation.