A cDNA clone encoding cytochrome P-450(11)beta of rat adrenal has been cloned and sequenced using a bovine P-450(11)beta cDNA insert (pcP-450(11 beta)-2; (1987) J. Biochem. 102, 559-568) as a probe. The nucleotide sequence contains an open reading frame sufficient to encode the entire amino acid sequence of a P-450(11)beta precursor protein consisting of 499 amino acids including an extension peptide of 24 amino acids at the NH2-terminus. The cDNA contains 1247 nucleotides at the 3'-noncoding region including 51 nucleotides of poly A, but lacks the 5'-noncoding region. The deduced amino acid sequence shows 61% similarity to that of bovine P-450(11)beta. Putative binding sites for heme and steroid are highly conserved among steroidogenic P-450s of known structure.