The effectiveness of single vs. separate pulses of low doses of free estradiol on the induction of sexual receptivity in ovariectomized female rats was examined. Two separate subcutaneous pulses of low doses of free estradiol at 0 and 6 hours induced high levels of progesterone facilitated receptivity at 24 hours. However, a single pulse of the same total dose of estradiol induced little or no receptivity at 24 hours (or at 18 hours). In fact, approximately 10 times as much estradiol was required in a single pulse to induce comparable receptivity. The smaller behavioral response to a single pulse was not likely due to a ceiling effect or an unutilized excess of the hormone after one injection. This suggestion is based on measurements of the redistribution of estrogen receptors from the cytoplasm to cell nuclei as an index of an initial cellular stimulus. Doses of estradiol used in the behavioral studies included several doses that were subsaturating with regard to hypothalamic estrogen receptors. The greatly enhanced behavioral response to two separated pulses of estradiol is consistent with the hypothesis that an initial action of estradiol may be to sensitize target tissues to further stimulation by the hormone.