Methyltrienolone, a synthetic steroid, was used as a photoaffinity ligand for steroid-binding proteins. The enzymatic activity of bovine adrenocortical cytochrome P-450 11β was inhibited by methyltrienolone in a competitive manner without exposure to light and cytochrome P-450 11β was photolabeled with methyltrienolone after irradiation with UV light. The addition of 11-deoxycorticosterone during photolabeling protected cytochrome P-450 11β from photolabeling. Photolabeled cytochrome P-450 11β was digested with TPCK-treated trypsin and the peptide fragments were separated with a reverse-phase HPLC system. The labeled peptide was analyzed and its amino acid sequence was determined to be Trp 428-Leu 429-Asp 430-Arg 431. Alignment of the primary structure of cytochrome P-450 11β with that of cytochrome P-450 cam revealed that the identified sequence corresponds to the region between the β3-sheet and L-helix of cytochrome P-450 cam . This region of mammalian cytochromes P-450 shows poor homology with that of cytochrome P-450 cam , but is well-conserved, especially at Trp-428 and preceding amino acids, as the aromatic region. The present results demonstrate that the labeled sequence contributes in part to the formation of the substrate binding pocket of cytochrome P-450 11β which was not expected from the results of the primary sequence alignment with cytochrome P-450 cam .