Corticosteroids inhibit ACTH secretion through diverse cellular mechanisms, including direct pituitary and indirect suprapituitary effects. Although exogenous CRH provides a useful assessment of corticotroph function, the suprapituitary component of ACTH regulation has been difficult to assess in humans. Naloxone (NAL) has been reported to stimulate ACTH secretion indirectly through the release of endogenous hypothalamic CRH, suggesting its potential application in the examination of suprapituitary regulation of ACTH secretory dynamics. We sought to examine the inhibitory effects of corticosteroids on kinetic parameters of ACTH secretion, assessed by a deconvolution method, in healthy human subjects. We also sought to directly compare the ACTH responses to serial administration of human CRH and NAL as well as spontaneously occurring ACTH pulses to distinguish pituitary and suprapituitary components of hypothalamic-pituitary-adrenal regulation. Normal healthy subjects (n = 11) received hCRH (0.4 microgram/kg) at 1800 h and then NAL (65 micrograms/kg) at 1930 h, respectively, on 3 separate study days: placebo pretreatment plus CRH/NAL stimulation, metyrapone (MET) pretreatment plus CRH/NAL, or MET alone. Plasma ACTH and serum cortisol were assessed at frequent (every 10 min) intervals during CRH/NAL or placebo infusions (1800-2100 h) on all 3 study days, and deconvolution analysis was performed to determine kinetic parameters of endogenous and stimulated ACTH secretion. Suppression of endogenous cortisol secretion with MET significantly increased both continuous (basal secretion rate) and pulsatile CRH- and NAL-stimulated ACTH bursts (P < 0.05). The increase in total ACTH secreted per burst was related to two distinct effects of cortisol regulating the amplitude (maximum secretion rate) and half-duration (P < 0.05) of secretory bursts. The ACTH responses to CRH and NAL for individual subjects were significantly and positively correlated in both placebo pretreatment plus CRH/NAL stimulation and MET pretreatment plus CRH/NAL studies (P < 0.01). MET administration disproportionately increased the ACTH response to NAL, producing a significant increase (P < 0.01) in the slope of the regression relating ACTH responses to CRH and NAL. The following conclusions were made: 1) endogenous cortisol secretion, even at levels associated with relatively low serum cortisol concentrations, exerts a significant negative feedback effect on both continuous and pulsatile ACTH secretion; 2) cortisol inhibits pulsatile ACTH secretion through distinct regulatory mechanisms that independently modulate both the mass and the duration of ACTH secretory bursts; 3) the differential sensitivity of the CRH- and NAL-stimulated ACTH responses to MET administration suggests that both pituitary and suprapituitary components of the hypothalmic-pituitary-adrenal axis are sensitive to negative regulation over a rapid or intermediate temporal domain. Endogenous cortisol modulates multiple components of dynamical ACTH secretion through composite effects that appear to be mediated through structurally and functionally distinct regulatory domains.