Cytochrome P450 (P450)-halothane complex formation, an index of reductive metabolism of halothane, was investigated by using rat hepatic microsomes and purified rat P450s under anaerobic conditions. P450-halothane complex formation was produced by the hepatic microsomes from phenobarbital and dexamethasone treated rats. Anti-P450 3A2 and 2B1/2 antibodies extensively inhibited complex formation in hepatic microsomes from dexamethasone and phenobarbital treated rats, respectively. In reconstituted systems using purified rat P450s, P450 3A2 and 2B1 complexed halothane efficiently. Complex formation was also recognized in human hepatic microsomes under anaerobic conditions.