The effect of gonadectomy and subsequent gonadal steroid administration on hypothalamic corticotropin-releasing factor immunoreactivity (CRF-ir) in male and female rats was determined using a radioimmunoassay specific for rat CRF. When compared to appropriate controls, gonadectomy resulted in significantly decreased CRF-ir in both males and females (p less than 0.025). Short-term replacement with testosterone or estradiol did not significantly alter hypothalamic CRF-ir. Acute IC administration of gonadotropin-releasing hormone to male rats significantly decreased CRF-ir in the median eminence 15 minutes later. Chronic administration of estradiol for 3 weeks significantly reduced CRF-ir in ovariectomized rats (p less than 0.005). This decrease was shown not to correlate to the marked increase in anterior pituitary weight (r2 = 0.063, not significant). Chronic administration of estradiol to hypophysectomized female rats significantly decreased hypothalamic CRF-ir (p less than 0.01), an effect localized to the median eminence (p less than 0.005). These data imply that the gonadectomy-induced reduction in hypothalamic CRF-ir was not directly mediated by the loss of gonadal steroids. Chronic estradiol treatment decreased hypothalamic CRF-ir, by a non-pituitary mechanism. These data show significant effects of the gonadal axis in the regulation of CRF in the hypothalamus in both sexes.