The regulation of GTP-cyclohydrolase (GTP-CH) activity and tetrahydrobiopterin (BH4) levels in the adrenal cortex were studied in intact and hypophysectomized rats. Treatment with a single dose of reserpine (5 mg/kg) or insulin-induced hypoglycemia (4 h) elevated adrenocortical BH4 3-fold by 10 h; BH4 levels remained elevated after 24 h and returned to control levels by 48-72 h. GTP-CH was significantly increased 24 h after hypoglycemic shock, and the increased enzyme activity preceded the changes in BH4 levels after reserpine treatment. Two and a half hours of stress by immobilization also increased GTP-CH activity and BH4 levels in the adrenal cortex. The activities of sepiapterin reductase and dihydrofolate reductase, putative enzymes in the biosynthetic pathway from GTP to BH4, were not increased by reserpine. Both reserpine and insulin increased the apparent maximum velocity for GTP, with no increase in the affinity of the enzyme for its substrate, further suggesting that the experimental treatments induce the synthesis of GTP-CH. Hypophysectomy completely blocked the reserpine-dependent increase in both cortical GTP-CH activity and BH4 content. The administration of purified porcine ACTH to intact and hypophysectomized rats elevated adrenocortical GTP-CH activity and cofactor levels. Synthetic ACTH-(1-24) also enhanced the enzyme activity and BH4 levels in the adrenal cortex. Thus, pituitary control of adrenal cortical GTP-CH synthesis and biopterin levels appears to be mediated through the secretion of ACTH. The changes in enzyme activity and cofactor levels after activation of the hypothalamo-hypophyseal axis or ACTH administration suggest that BH4, a cofactor for certain monooxygenases, has some function, as yet unknown, in the adaptive changes of the adrenal cortex in response to stress.