The development of the rat adrenal gland during late gestational and neonatal stages was studied by following the expression of aldosterone synthase cytochrome P450 (P450aldo) and glucocorticoid-synthesizing cytochrome P450 (P45011 beta). Cells expressing P450aldo, a functional marker for the mineralocorticoid-synthesizing zona glomerulosa, were not detected until day 20 of fetal age, i.e., 2 days before birth, although the zona glomerulosa cells were histologically recognizable at the 18th day of gestation. The intensity of P450aldo staining thereafter became stronger with age in the outer portion of the cortex. Cells expressing P45011 beta, a marker for the glucocorticoid-producing zona fasciculata, were present in the fetal adrenals on the 18th day. P45011 beta-positive cells were distributed over the whole adrenal gland and intermingled with the cells containing tyrosine hydroxylase, a marker enzyme for medullary cells. The P45011 beta-positive and tyrosine hydroxylase-positive cells began to separate on the 20th day, and were completely resolved from each other around the third day after birth. Expression of P450aldo and P45011 beta, together with that of tyrosine hydroxylase, thus serves as a suitable marker for studying the development of the adrenal gland.