A cDNA for cytochrome P-450(11 beta,aldo) was cloned from a library of bullfrog interrenal tissue (tissue corresponding to the mammalian adrenal gland). The 1919-bp cDNA encoded a protein of 517 amino acids. Its amino acid sequence was highly similar to the sequences of bovine P-450(11 beta) and rat P-450(11 beta,aldo) when P-450(11 beta) family enzymes reported to date were examined. The enzyme expressed in COS7 cells had the 11 beta-hydroxylation, 18-hydroxylation activities and aldosterone synthetic activity. Northern-blot and immunoblot analyses suggested that a single P-450(11 beta) enzyme was expressed in bullfrog interrenal tissue. These results suggest that a single enzyme catalyzes the final steps of glucocorticoid and mineralocorticoid biosynthesis in bullfrog interrenal tissue as in bovine adrenal gland. A phylogenetic tree of CYP11B genes suggests that the frog enzyme diverged at an earlier evolutionary time from other vertebrate enzymes. Immunohistochemical and in situ hybridization studies indicated that steroidogenic cells existed in the outer region of interrenal tissue more densely than in the inner region, whereas some medullary cells made clusters like islets. Most of the cells were diffusely distributed in the tissue.