12alpha-Hydroxylation of 5beta-cholestane-3alpha, 7alpha-diol was studied in reconstituted systems consisting of electrophoretically homogeneous cytochrome P-450 LM4 fractions and NADPH-cytochrome P-450 reductase from rabbit liver microsomes. Cytochrome P-450 LM4 fractions were prepared from untreated, phenobarbital-treated, beta-naphthoflavone-treated and starved rabbits. The purified cytochromes catalyzed 12alpha-hydroxylation more efficiently than the corresponding microsomes. In the reconstituted systems, carbon monoxide inhibited 12alpha-hydroxylation by 50-80%. The rate of 12alpha-hydroxylation was three to four times higher with cytochrome P-450 LM4 fractions from starved rabbits than with cytochrome P-450 LM4 fractions from untreated, phenobarbital-treated and beta-naphthoflavone-treated animals. Amino acid analyses, peptide mapping experiments as well as absorption spectral and circular dichroism spectral analyses revealed physical differences between cytochrome P-450 LM4 fractions from starved animals and preparations from phenobarbital-treated animals. The results indicate the presence of a cytochrome P-450 species in the cytochrome P-450 LM4 fraction specific for 12alpha-hydroxylation.