The mechanism by which protein malnutrition increases circulating glucocorticoids is unclear. To determine whether ACTH synthesis and secretion also increase in protein malnutrition, rats were sham adrenalectomized or adrenalectomized and replaced with varying amounts of corticosterone before dietary protein deprivation. Pair-fed rats served as controls for reduced voluntary food intake in protein-deprived rats. Dietary protein deficiency, but not pair-feeding, increased resting plasma corticosterone in sham-adrenalectomized rats. Restraint-induced ACTH secretion was not inhibited by the increased basal corticosterone levels in protein-deficient rats. When increases in corticosterone were eliminated by adrenalectomy or controlled by adrenalectomy with low level corticosterone replacement, increases in resting plasma ACTH and anterior pituitary POMC messenger RNA expression occurred with protein deprivation that could be statistically discriminated by regression analysis from changes due to caloric restriction (pair-feeding) and overt glucocorticoid feedback resistance. We conclude that protein malnutrition increases pituitary-adrenocortical activity at least in part by specifically increasing the drive for ACTH synthesis and secretion.