Previous studies have shown that sc administration of androgen or estrogen into newborn female rats will disturb postpuberal ovulatory function, as evidenced by a higher incidence of vaginal estrus, few ovarian corpora lutea, and the abolition of estrogen-induced luteinizing hormone surges after ovariectomy and chronic estrogen maintenance. Studies of the uptake of labeled estrogen and intracerebral lesion studies have indicated the preoptic area and anterior hypothalamic nuclei (POA-AH) as possible sites of steroid uptake and control of certain sexual functions in the rat. Attempts to localize the site of steroid effects to the POA-AH of newborn rats by chronic implants have been hampered by lack of definition of extent of localization in space and time. The present study introduces a microinjection technique for the delivery of subpicogram quantities of estradiol to these areas of the central nervous system in newborn rats, and statistically defines the extent of steroid localization in three dimensions as well as the duration of exposure. Delivery of estradiol by this method in the POA-AH region yielded an increased incidence of postpuberal vaginal estrus; a decrease in the number of corpora lutea; and in many but not all, led to abolition of estrogen-progesterone induced LH surges after castration and estrogen replacement. Injection 0.5 mm more caudad did not result in blockade of the estrous cycle.