Studies were carried out to define the mechanism of action of growth hormone [GH] on adrenocortical steroidogenesis in hypophysectomized female rats. ACTH administration for 7 days increased corticosterone secretion in vivo and corticosterone production by adrenal tissue in vitro. Adrenal mitochondrial and microsmal cytochrome P-450 concentrations and the activities of cytochrome P-450-dependent enzymes (cholesterol sidechain cleavage, 11.beta.-hydrolase, 21-hydroxylase) were also increased by ACTH. Administration of bovine GH alone to hypophysectomized rats had no effect on any of the parameters evaluated. When given in combination with ACTH, GH synergistically enhanced the effects of ACTH on cholesterol sidechain cleavage activity and corticosterone secretion. The magnitude of the pregnenolone-induced difference spectrum in adrenal mitochondria, indicative of cholesterol binding to cytochrome P-450 content nor the activities of other steroidogenic enzymes were affected. GH interacts with ACTH to promote corticosterone secretion by increasing the association of cholesterol with adrenal mitochondrial cytochrome P-450, thereby increasing the activity of cholesterol sidechain cleavage, the rate-limiting step in steroidogenesis.