We have isolated and characterized two isozymes of mouse steroid 11β-hydroxylase (11β-OHase), designated 11β-OHase and aldosterone synthase (AS). Physical mapping of overlapping cosmid and phage isolates defined two genes (designated Cyp11b-1 and Cyp11b-2 in the standard nomenclature for cytochrome P450 genes) that are oriented in the same direction and separated by approximately 8 kilobase pairs of DNA. The two genes are highly homologous in their coding regions, with 84% nucleotide identity and 86% predicted amino acid identity. In regions where the sequences of the rat 11β-OHase and AS genes diverged most widely, the mouse sequences also differed significantly, thereby identifying putative mouse 11β-OHase and AS genes. Both genes were mapped to chromosome 15 by analyzing restriction fragment length variations in a panel of DNA samples from an interspecific cross. To determine the functional properties of the 11β-OHase and AS proteins, we transfected COS-7 cells with plasmids that expressed the proteins encoded by the 11β-OHase and AS genes. When expressed in transfected COS-7 cells, the 11β-OHase protein converted deoxycorticosterone to corticosterone but did not produce aldosterone. Consistent with its postulated role in mineralocorticoid biosynthesis, the product of the AS gene efficiently synthesized aldosterone. We next studied the expression of these two isozymes in Y1 adrenocortical tumor cells and in the intact mouse adrenal gland. Although Y1 cells otherwise resemble zona fasciculata cells and express the 11β-OHase gene at high levels, transcripts encoded by the AS gene were detected at levels approximately 10-fold lower than the 11β-OHase transcripts. In situ hybridizations of adrenal sections with gene-specific probes showed that the 11β-OHase gene was expressed in the zona fasciculata, whereas AS-derived transcripts were detected only in the outer zona glomerulosa. This selective expression of the mouse 11β-OHase isozymes in different adrenocortical zones very likely contributes significantly to its functional separation into glucocorticoid- and mineralocorticoid-producing compartments. © 1991 by The Endocrine Society.