Secretion of cortisol, corticosterone, and aldosterone was measured in vivo in normal and sodium-depleted hypophysectomized dogs. Biogenesis of steroids was then measured in vitro with outer slices of the adrenals of the same dogs. In some studies, metyrapone or puromycin was added. In vivo, sodium depletion stimulated the production of cortisol, corticosterone, and aldosterone. In vitro, tissues from sodium-depleted animals released more aldosterone, but less corticosterone than those from sodium-replete controls. The results are interpreted to indicate that (a) biosynthesis of aldosterone is regulated at at least two sites in the biosynthetic pathway. The final conversion, that of corticosterone to aldosterone, is stimulated by sodium depletion. This effect persists for at least 3 hr while slices from sodium-depleted dogs are incubated in vitro. Stimulation at this site is thus relatively stable in vitro; its activation by sodium depletion is not inhibited by puromycin in the dog. Stimulation at this site can explain, at least in part, the increased effectiveness of adrenocorticotropin (ACTH) on aldosterone biogenesis during sodium depletion.(b) the earlier site at which sodium depletion stimulates the secretion of aldosterone is "above" the position of desoxycorticosterone in the pathway; it is probably at the conversion of cholesterol to pregnenolone. Stimulation at this site is quickly lost during incubation of adrenal slices. It is thus relatively unstable in vitro; its activation by sodium depletion is inhibited by puromycin in the dog.