Two forms of cytochrome P-450 with different substrate specificities were isolated from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A specific antibody was produced toward the major form of the cytochrome. The antibody inhibits microsomal acetanilide hydroxylation (80%). It does not cross-react with the minor fraction of the cytochrome or inhibit the hydroxylation of 3,4-benzpyrene or coumarin, the N-demethylation of aminopyrine or the O-deethylation of 7-ethoxycoumarin catalyzed by rabbit liver microsomes. The major form has an estimated Mr = 54,000 and displays an n-octylamine difference spectrum with an absorption maximum at 426 nm and a minimum at 391 nm. When reconstituted, this cytochrome catalyzes acetanilide hydroxylation at a higher rate than microsomes or the minor fraction. The n-octylamine difference spectrum of the minor fraction displays an absorption maximum at 431 nm and a minimum at 410 nm. When reconstituted, this fraction catalyzes the hydroxylation of 3,4-benzpyrene and the O-deethylation of 7-ethoxycoumarin. The two cytochromes appear to be distinct entities and function in different catalytic pathways.