Incubation of halothane with rabbit liver microsomes in the presence of NADPH and oxygen resulted in the formation of trifluoroacetic acid (TFAA). No TFAA was detected in the absence of either NADPH or oxygen. TFAA also was found when a 9000 x g supernatant fraction (with NADPH and oxygen) was used instead of the microsomal fraction. The 105,000 x g supernatant fraction (with NADPH and oxygen), however, did not produce detectable levels of TFAA. Phenobarbital pretreatment of the animals resulted ina significant increase in both the formation of TFAA and the cytochrome P-450 content of microsomes. A good correlation was found between formation of TFAA and microsomal cytochrome P-450 levels. The amount of TFAA formed in hepatic microsomes was significantly inhibited by carbon monoxide (CO), SKF 525-A (2-diethylaminoethyl-2,2,-diphenylvalerate HCl) and p-chloromercuribenzoic acid. On the other hand, TFAA was formed when NADH was used as an electron donor instead of NADPH. In conclusion, these results clearly demonstrate the metabolism of halothane by the hepatic microsomal mixed-function oxidase system.