In an attempt to obtain a more precise localization of the neural target site(s) of estrogen-priming action in the activation of estrous behavior, 30-gauge cannulae containing a preparation of 17 beta-estradiol diluted 1:250 with cholesterol were implanted bilaterally into the brains of ovariectomized rats. Subjects were tested for sexual behavior with intact males at 4-day intervals for 3 weeks beginning 2-3 days after stereotaxic surgery. Animals received a systemic injection of 0.5 mg progesterone 4-6 h before all but 1 test. After this treatment, estrous behavior was observed in 19 of the 20 animals judged to have both cannulae resting in or within 0.25 mm of the ventromedial nucleus of the hypothalamus (VMN). Virtually no estrous behavior was observed without the administration of progesterone. Behavioral scores decreased as a function of the distance of the cannulae tips from the VMN. Cholesterol implants in the same region (n = 8) were without effect. Implants in other regions of the brain (e.g. preoptic area, diagonal band of Broca, lateral habenula, amygdala, or cortex; n = 52) were ineffective. These results indicate that estrogenic stimulation of the region of the VMN alone is sufficient to prime the activation of estrous behavior in the ovariectomized rat.