How sex steroids modulate glucocorticoid feedback on the hypothalamic-pituitary-corticotrope (HPC) unit is controversial in humans. We postulated that testosterone (T) in men and estradiol (E₂) in women govern unstressed cortisol-mediated negative feedback on ACTH secretion. To test this hypothesis, 24 men and 24 women age 58 ± 2.4 yr were pre-treated with leuprolide and either sex steroid (E₂ in women, T in men) or placebo addback. Placebo or ketoconazole (KTCZ) was administered overnight to inhibit adrenal steroidogenesis during overnight 14-h i.v. infusions of saline or cortisol in a continuous vs pulsatile manner to test for feedback differences. ACTH was measured every 10 min during the last 8 hr of the infusions. The main outcome measures were mean ACTH concentrations, pulsatile ACTH secretion and ACTH ApEn (approximate entropy). ACTH concentrations were lower in women than men (P<0.01), and in women in the E₂ + compared with E₂ - group under both continuous (P=0.01) and pulsatile (P=0.006) cortisol feedback, despite higher CBG and lower free cortisol levels in women than men (P<0.01). In the combined groups, under both modes of cortisol addback, ACTH concentrations, pulsatile ACTH secretion and ACTH secretory-burst mass correlated negatively and univariately with E₂ levels (each P<0.005). E₂ also suppressed ACTH ApEn (process randomness) during continuous cortisol feedback (P=0.004). T had no univariate effect but was a positive correlate of ACTH when assessed jointly with E₂ (negative) under cortisol pulses. In conclusion, sex steroids modulate selective gender-related HPA-axis adaptations to cortisol feedback in unstressed humans.