Steroidogenic factor-1 (SF-1), has emerged as a critical nuclear receptor regulating development and differentiation at several levels of the hypothalamic-pituitary-steroidogenic axis. Although many coregulatory factors have been shown to physically and functionally interact with SF-1, the relative importance of these interactions in SF-1 target tissues has not been thoroughly established. In this study we assessed roles of steroid receptor coactivator-1 (SRC-1) in hypothalamic-pituitary-adrenal (HPA) axis function using SRC-1-deficient (SRC-1-/-) mice in the absence or presence of SF-1 haploinsufficiency. Surprisingly, SRC-1 deficiency did not alter baseline HPA axis function or the acute rise in corticosterone after ACTH administration and failed to exacerbate adrenocortical dysfunction in SF-1+/- mice. However, after exposure to paradigms of acute and chronic stress, SRC-1-/- mice exhibited an elevation in serum corticosterone despite normal (nonsuppressed) ACTH, suggesting an increase in adrenal sensitivity as well as a concomitant defect in glucocorticoid-mediated feedback inhibition of the HPA axis. An examination of potential compensatory mechanism(s) revealed an increase in adrenal weight, selective elevation of melanocortin 2 receptor mRNA, and a coincident increase in SRC-2 and SRC-3 expression in SRC-1-/- adrenals. A reduction in blood glucose was observed in SRC-1-/- mice after chronic stress, consistent with a generalized state of glucocorticoid resistance. Dexamethasone suppression tests confirmed a weakened ability of glucocorticoids to 1) elevate serum glucose levels and induce hepatic phosphoenolpyruvate carboxykinase transcription and 2) suppress pituitary proopiomelanocortin transcript levels in SRC-1-/- animals. Collectively, these data are consistent with an indispensable role for SRC-1 in mediating actions of glucocorticoids in pituitary and liver.