Halothane-induced lipid peroxidation was studied in microsomes from phenobarbital-pretreated male rats at defined steady state oxygen partial pressures (Po·2). At Po·2less than 10 mmHg on addition of halothane to NADPH-reduced microsomes, significant increases in malondialdehyde (MDA) formation, oxygen uptake, and conjugated dienes were measured. At the maximum, near a Po2of 1 mmHg, halothane induced the formation of about 0.75 nmol MDA · mg microsomal protein−1· min−1; it also stimulated microsomal oxygen uptake twofold to threefold, and caused an almost threefold increase in conjugated diene absorption. Moreover, at this Po2, microsomal glucose-6-phosphatase lost about 70% of its activity. At Po2greater than 10 mmHg, no significant effects of halothane on MDA formation, oxygen uptake, conjugated diene absorption, and glucose-6-phosphatase activity were observed; likewise under anaerobic conditions there was only a slight increase in conjugated dienes. The findings demonstrate that halothane induces microsomal lipid peroxidation at low Po2and in the presence of particular cytochrome P-450 isoenzymes, and that the halothane-induced lipid peroxidation leads to severe microsomal lesions, as indicated by the loss of glucose-6-phosphatase activity.