Oxidation of rat hepatic tissue was measured as incorporation of oxygen-18 (18O) from 18O2 following exposure to carbon tetrachloride (CCl4). Anesthetized rats were injected with CCl4 and allowed to breathe 18O2 for 1 hr, and then livers were homogenized, fractionated, and dried. The dried fractions were pyrolyzed to CO by an oxygen elemental analyzer, the CO was oxidized to CO2, and isotope ratio mass spectrometry was used to determine the abundance of 18O in the CO2. Rats that breathed 18O2 (21% in N2) for 1 hr had significant incorporation of 18O into lipids, solutes, and macromolecules of the liver. Injection with CCl4 increased incorporation of 18O into all liver fractions, although this increase was significant only in the lipid fraction. Rats pretreated with phenobarbital and then given CCl4 had significantly increased 18O in all liver fractions although it was greatest in the lipid fraction. About 5 mumol of 18O per gram of dry liver was incorporated in phenobarbital/CCl4-treated rats, of which 60% was in the water-soluble fraction, 17% in the lipids, and 16% in the macromolecules. Piperonyl butoxide administration abolished the CCl4-induced 18O incorporation. Thus, 18O incorporation appeared to provide a measure of oxidation in all tissue fractions following in vivo CCl4-induced liver injury.