In situ hybridization histochemistry was used to monitor the expression of 3β-hydroxysteroid dehydrogenase, Δ4-isomerase (3βHSD) and cytochrome P450 11β-hydroxylase (P45011β) messenger RNA (mRNA) in adult rat adrenals after stimulation in vivo. In Exp 1, adrenals were collected from rats injected with saline or ACTH for 1, 2, 3, or 4 days. Adrenal sections from saline-treated rats showed uniform expression of 3βHSD mRNA that extended from the adrenal capsule to the medullary border. In contrast, P45011β mRNA showed high levels in the outer fasciculata and low levels in the inner fasciculata/reticularis. In response to ACTH, the integrated density of 3βHSD hybridization did not increase until 4 days. The integrated density of P45011β hybridization increased in ACTH-treated rats between 1-4 days due to increased hybridization in the inner fasciculata/reticularis. In Exp 2, rats were treated with ACTH or saline, and adrenals were harvested at 4, 8, or 24 h. The hybridization density of 3βHSD did not change after ACTH or saline injection. Increased expression of P45011β mRNA was observed at 4 and 8 h, but not 24 h post-ACTH. In Exp 3, to determine the response to acute stress, adrenals were collected from rats 24 h after surgical laparotomy. The integrated density of 3βHSD labeling did not change, whereas both hybridization area and mean density of P45011β increased. Increased expression of P45011β mRNA was observed in the inner fasciculata similar to that observed after ACTH injection. In addition, adrenal cells were more responsive to ACTH in vitro after surgical stress. These results suggest that the rat adrenal cortex can respond to acute stress by up-regulation of the expression of steroidogenic enzyme genes and that this occurs in part by increasing the number of cells actively expressing P45011β mRNA. The adrenal response after stress most likely results at least in part from stimulation by ACTH. These findings suggest that changes in adrenal steroidogenesis in response to ACTH may result from recruitment of steroidogenic cells to synthesize and secrete corticosteroids.