Three experiments were conducted to evaluate negative feedback action of estradiol-17 beta (E2) on luteinizing hormone (LH) release in ovariectomized heifers and to determine the response at different ages. In the first experiment, iv injections of .2 or 2.0 micrograms E2/kg body weight (BW) reduced serum concentrations of LH by arresting episodic mode of secretion. Results of a second experiment indicated a positive relationship (P less than .05) between dose of E2 and duration of LH suppression. Mean duration of suppression with the high (2.0 micrograms E2/kg BW), medium (2 micrograms E2/kg BW) and low (.1 microgram E2/kg BW) doses was 6.5, 4.5 and 3.5 h, respectively. Neither magnitude nor rate of LH suppression was related (P less than .25) to dose in the first two experiments. Results of third experiment of showed that both magnitude and duration of suppression were directly proportional to dose of E2 (0, .1 and 1.0 microgram E2/kg BW). The response lasted longer in heifers 4 mo of age than in heifers 8 and 12 mo of age. E2 was also more effective in reducing LH pulses in heifers 4 mo of age than in heifers 8 and 12 mo of age. These results show that E2 inhibits LH release by influencing pulsatile mode of secretion, and that the threshold to negative feedback increases as heifers approach puberty.