A cDNA clone encoding the complete protein sequence of the precursor form of bovine cytochrome P450(11)beta has been constructed using a combined technique of first strand cDNA synthesis by reverse transcription followed by polymerase chain reaction. Upon expression of this cDNA in COS 1 cells the P450(11)beta is found to be proteolytically processed and localized in the mitochondrion. This cDNA encodes the major form of P450(11)beta found in bovine adrenal cortex (designated 11 beta-3; Kirita, S., Morohashi, K., Hashimoto, T., Yoshioka, H., Fujii-Kuriyama, Y., and Omura, T. (1988) J. Biochem. 104, 683-686) and is capable of catalyzing 11 beta-hydroxylation of deoxycorticosterone, 11-deoxycortisol, and androstenedione in COS 1 cells as well as aldosterone synthesis from deoxycorticosterone. In addition, a second form of P450(11)beta (herein designated 11 beta-4), having no detectable 11 beta-hydroxylase activity or aldosterone synthase activity, was found in the local bovine population by this cloning procedure. These two forms of P450(11)beta (11 beta-3 and 11 beta-4) contain five amino acid differences between them, all located within the amino-terminal half of the molecules. By changing 2 of the amino acids in the inactive form to the corresponding amino acids in the active form (Leu66----Phe and Pro126----Ser) both 11 beta-hydroxylase and aldosterone synthetase activities were completely restored. Neither of these changes alone led to detectable activity. Thus, upon expression in mitochondria of heterologous cells, bovine P450(11)beta catalyzes both 11 beta-hydroxylation and aldosterone synthesis as reported previously for the purified enzyme in an in vitro reconstituted system, and Phe66 and Ser126 seem to be important residues in maintaining both activities.