Two homologs of the gene encoding the adrenocortical 21-hydroxylase (21-OHase) are located within the S region of the mouse major histocompatibility complex. Only one of these homologs, however, encoded the full-length sequence of 21-OHase, directed the synthesis of 21-OHase RNA in the mouse adrenal gland, and was capable of restoring 21-OHase activity when transfected into 21-OHase-deficient Y1 adrenocortical tumor cells. Y1 cells transfected with the 21-OHase gene, when stimulated with ACTH, increased the number of 21-OHase transcripts up to 10-fold. The 21-OHase gene was not expressed when transfected into mouse fibroblast L cells, and was poorly expressed and poorly regulated by ACTH when transfected into a Y1 mutant harboring a defective cAMP-dependent protein kinase. Marked decreases in expression of the 21-OHase gene were noted when DNA constructs that contained fewer than 230 base pairs in the 5' flanking region of the gene were transfected into Y1 cells. These results indicate that the 21-OHase gene encodes information required for the tissue-specific expression and hormonal regulation of 21-OHase. The cAMP-dependent protein kinase is important for both aspects of gene expression. At most, 230 base pairs of 5' non-coding information are required for efficient expression of the 21-OHase gene in Y1 cells. © 1986 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.