Macrophage migration inhibitory factor (MIF) is a proinflammatory pituitary and immune cell cytokine and a critical mediator of septic shock. It has been reported that MIF is secreted in parallel with ACTH from the pituitary in response to stress or inflammatory stimuli. MIF release from immune cells is also induced rather than inhibited by glucocorticoids. It has therefore been suggested that MIF may be a novel counter-regulatory hormone of glucocorticoid action that acts both as a paracrine and endocrine modulator of host responses. We have measured circulating MIF levels, using a human MIF ELISA, in normal subjects and patients under numerous pathophysiological conditions. Serum MIF was measured in normal subjects who underwent stimulation of the hypothalamo-pituitary-adrenal axis with an insulin tolerance test (n = 8), a CRH-stimulation test (n = 5), a short synacthen test (n = 5), and following a low-dose dexamethasone suppression test (n = 6). We also sampled from a peripheral vein and both inferior petrosal sinuses before and after CRH stimulation in four patients with a histologically proven diagnosis of Cushing's disease. Immunostaining of the pituitary tumors for MIF was also performed. In normal subjects serum MIF levels did not rise in parallel with cortisol during the insulin tolerance or CRH test or after administration of synthetic ACTH. In all subjects cortisol levels became undetectable after the low-dose dexamethasone suppression test, and no consistent change was observed in serum MIF levels during the test. In patients with Cushing's disease, there was no basal central-to-peripheral gradient in MIF, and no consistent changes occurred in serum MIF levels in either the left or right inferior petrosal sinus after CRH stimulation; however, immunostaining of the surgically removed pituitary tumors from the same patients showed strong staining for both ACTH and MIF. These results show that in humans acute modulation of the hypothalamo-pituitary-adrenal axis does not significantly alter circulating MIF levels. In addition, ACTH-secreting pituitary tumors that express MIF do not release MIF either spontaneously or in response to CRH stimulation, and there is no gradient for MIF in the venous drainage of the pituitary. Our study suggests that the pituitary gland is not the major contributor to circulating MIF; an autocrine or paracrine role for pituitary-derived MIF is more likely.