A stratum of cells that did not contain both aldosterone synthase cytochrome P450 (cytochrome P450aldo) and cytochrome P45011 beta was found immunohistochemically between the zona glomerulosa and the zona fasciculata of the rat adrenal cortex. As cytochromes P450aldo and P45011 beta are the enzymes responsible for the biosynthesis of aldosterone and corticosterone, respectively, the cells there are considered to be incapable of synthesizing both aldosterone and corticosterone. Furthermore, the cells are regarded as inert in producing adrenal androgens, because rat adrenal cortex is known to lack steroid 17 alpha-hydroxylase. Thus, the stratum is composed of cells that do not synthesize any of the major corticosteroids in significant quantities. It was 5-10 cells thick under normal feeding conditions, but diminished to 4-5 cells thick when animals were maintained under Na restriction, which is known to stimulate the secretion of angiotensin-II. When the distribution of 5-bromo-2'-deoxyuridine-labeled nuclei in the adrenocortex from BrdU-administered rats was examined, the stained nuclei were concentrated in and around the cell stratum. The pulse-chase experiments showed that the labeled cells migrated out of this layer and into the zonae fasciculata-reticularis. On the basis of these findings, we suggest that the newly discovered cell layer is the progenitor cell zone of the rat adrenal cortex.