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A recombinant cDNA clone, PBC21-1, specific for bovine steroid 21-hydroxylase cytochrome P-450 (P-450C21) was identified in a bovine adrenocortical cDNA library, and this identity was confirmed by nucleotide sequencing which revealed significant amino acid homology (77%) with human P-450C21 cDNA. The pBC21-1 insert is 1.7 kilobases in length and includes a 1128 base pair region that encodes the C-terminal 376 amino acids of bovine P-450C21 as well as 535 base pairs of 3'-untranslated sequence. A novel feature of this insert is a 20 base pair intervening sequence near the 5' end, apparently the result of an aberrant splicing event. Northern blot analysis reveals that bovine P-450C21 is encoded by two transcripts, 2.3 and 2.0 kilobases in length which are detected in adrenal cortical RNA. Bovine liver, heart, kidney, and corpus luteum do not contain detectable P-450C21 transcripts. Regulation of P-450C21 gene expression by adrenocorticotropin was investigated with pBC21-1 and bovine adrenocortical cells in primary, monolayer culture. Treatment with ACTH or analogues of cAMP increases the steady-state levels of P-450C21 RNA in such cell cultures. In vitro transcription run-on assays suggest that this increase is, at least in part, due to the enhanced transcriptional activity of the P-450C21 gene.