The gene encoding barbiturate-inducible cytochrome P-450 BM-1 from Bacillus megaterium ATCC 14581 has been cloned and sequenced. An open reading frame in the 1.9 kb of cloned DNA correctly predicted the NH 2-terminal sequence of P-450 BM-1 previously determined by protein sequencing, and, in toto, predicted a polypeptide of 410 amino acid residues with an M r of 47 439. The sequence is most, but less than 27%, similar to that of P-450 CAM from Pseudomonas putida, so that P-450 BM-1 clearly belongs to a new P-450-gene family, distinct especially from that of the P-450 domain of P-450 BM-3, a barbiturate-inducible single polypeptide cytochrome P-450:NADPH- P-450 reductase from the same strain of B. megaterium (Ruettinger, R.T., Wen, L.-P. and Fulco, A.J. (1989) J. Biol. Chem. 264, 10987–10995).