I. Chloramphenicol (CAP) is a potent and selective mechanism-based inactivator of the major phenobarbital (PB)-inducible form of dog liver cytochrome P-450 (PBD-2) in vitro. In a reconstituted system, CAP inactivates PBD-2 in a time- and NADPH-dependent manner and binds covalently to the protein moiety of PBD-2 with a stoichiometry of 1 nmol CAP bound/nmol P-450 inactivated. In intact liver microsomes from PB-treated male Beagle dogs, CAP irreversibly inhibits androstenedione 16α- and 16β-hydroxylation and 2,4,5, 2',4',5'-hexachlorobiphenyl hydroxylation but not androstenedione 6 β -hydroxylation or NADPH-dependent triacetyloleandomycin (TAO) complex formation. Covalent binding of CAP to dog liver microsomes in vitro is increased 5.5-fold by PB induction. This increase correlates well with the increased levels of immunochemically determined PBD-2 (5.8-fold) and 16α - and 16β -hydroxylation of androstenedione (5.7- and 5.8-fold) in microsomes from PB-treated dogs. Anti-PBD-2 IgG significantly inhibits the covalent binding of CAP to microsomes from untreated and PB-treated dogs. Finally, CAP appears to bind covalently with a single protein with the same molecular weight as PBD-2, as evidenced by SDS-PAGE. II. A cytochrome P-450 called PBD-1 isolated from liver microsomes of an adult male Beagle dog treated with PB is structurally and functionally similar to members of the P450IIIA gene subfamily in rat and human liver microsomes. The sequence of the first 28 amino terminal residues of PBD-1 is identical in 15 and 20 positions, respectively, to the P450IIIA forms P450p from rat and P450(NF) from human. Upon immunoblot analysis, anti-PBD-1 IgG recognizes PCNa (P450p) and PCNb (PB\PCN-E) from rat, P450(NF) from human, and two proteins in liver microsomes from untreated and PB-treated dogs. Anti-PBD-1 IgG selectively inhibits P450IIIA form marker activities, including steroid 6β -hydroxylase, erythromycin demethylase and NADPH-dependent TAO complex formation in microsomes from PB-treated dogs. Major species differences exist in the apparent K(m) for 6β -hydroxylation of androstenedione by liver microsomes from humans, untreated rats and untreated dogs. In addition, evidence for functional heterogeneity of dog P450IIIA forms is presented: pretreatment of microsomes from PB-treated dogs with TAO plus NADPH had no effect on androstenedione 6β -hydroxylase activity.