Daily regeneration of rat adrenocortical cells were investigated in terms of circadian and zonal variations by following the cells at the DNA-synthesizing stage. An S-phase was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation into the cell-nuclei and/or by visualizing proliferating cell nuclear antigen. The BrdU-positive cells were observed throughout the day mainly in two regions of the adrenal cortex, i.e. the innermost portion of the zona glomerulosa and the outermost portion of the zona fasciculata. Cells only in a latter region showed a distinct circadian rhythm of cell proliferation with a peak at 3-4 a.m. A remarkable rise in the plasma adrenocorticotropin (ACTH) concentration preceded such an increase in the cell proliferation by about 4 hours. This phenomenon could be mimicked by raising the plasma ACTH concentration by the administration of Cortrosyn Z or metyrapone. Angiotensin II-stimuli induced by Na-deficiency increased the proliferation of zona glomerulosa cells in the former region at 6-7p.m without significant effects on that of the zona fasciculata cells in the latter region. Thus at least two sites, which respond differentially to the day/night cycle and circulating hormone levels, exist in rat adrenal cortex being responsible for the cytogenesis in this endocrine organ.