Gonadal steroids masculinize and defeminize neuroendocrine development, including behavior. Defeminization makes males less sensitive than females to estrogen for showing female sexual behavior and cyclic gonadotropin secretion. Masculinization makes males more sensitive than females to estrogen for showing male sexual behavior. Thus masculinization and defeminization produce opposite effects on estrogen sensitivity. To study the relationship between estrogen sensitivity and estrogen binding, we studied sex differences in estrogen binding to hypothalamic cell nuclei on a regional and temporal basis. We measured the amount of estradiol (E2) bound to cell nuclei in the preoptic area (POA), mediobasal hypothalamus (MBH), corticomedial amygdala, and cortex of gonadectomized male and female rats 30 and 60 min after [3H]E2 was injected intravenously. In the MBH, males consistently bound less E2 than females did. In the POA, males bound less E2 than females after 60 min, but they bound more E2 than females after 30 min. Decreased estrogen binding in the MBH may underlie defeminized sexual behavior. Similarly, decreased estrogen binding in the POA at 60 min may be a correlate of defeminized gonadotropin secretion, whereas increased estrogen binding in the POA at 30 min may be a correlate of masculinized sexual behavior. To test the hypothesis that decreased estrogen binding in the MBH and POA are correlates of defeminization, we measured E2 binding at 60 min in female rats in which masculinization and defeminization were manipulated independently. Defeminization decreased E2 binding to cell nuclei in both the POA and MBH to the level seen in males at this time point. Masculinization had no effect at this time point. The data suggest that sex differences in E2 binding to hypothalamic cell nuclei correlate reliably with sex differences in estrogen sensitivity even though masculinization and defeminization produce opposing effects on these parameters.