Estrogen binding was compared in cell nuclear KCl extracts from microdissected brain regions of gonadectomized-adrenalectomized male and female rats treated with a near-saturating dose of 17 beta-estradiol. Injection of 3.6 or 36.0 micrograms 17 beta-estradiol/kg BW, iv, 1 h before death resulted in a higher level of estrogen binding in the periventricular preoptic area (PVPOA), medial preoptic area, and ventromedial nucleus of the hypothalamus (VMN) of the female than in comparable tissue samples from the male. No significant sex differences in nuclear estrogen binding were observed in the arcuate-median eminence region, bed nucleus of the stria terminalis, or corticomedial amygdala. Scatchard analysis of saturation binding data revealed that the sex differences in cell nuclear estrogen binding in the PVPOA, medial preoptic area, and VMN reflect a difference in binding capacity rather than binding affinity. These in vitro biochemical findings were confirmed by autoradiographic studies. Gonadectomized-adrenalectomized animals were injected with 125I-labeled 11 beta-methoxy-16 alpha-iodoestradiol (2.0 micrograms/kg BW). Thin frozen sections (10 microns) through the preoptic area and hypothalamus were thaw-mounted onto microscope slides, then exposed against LKB Ultrofilm for 21 days. The autoradiographic images exhibited similar silver distributions and densities in males and females in the arcuate-median eminence region bed nucleus of the stria terminalis, and amygdala. However, 11 beta-[125I]methoxy-16 alpha-iodoestradiol uptake was lower in males than in females in the PVPOA and VMN. These results suggest that sex differences in responsiveness to estrogen stimulation in the rat may be due in part to sex differences in estrogen-binding capacity in specific regions of the hypothalamus that play important roles in the control of pituitary function and reproductive behaviors.