Ovariectomized rats were injected with estradiol benzoate (EB) on day 1. The rats received propylene glycol alone (control) or with Actinomycin-D (Act-D) at 08.00 h, followed by EB at 12.00 h on day 3. Plasma LH levels were markedly elevated at 18.00 h on days 3 and 4 in control rats; Act-D treatment failed to modify the surge of LH on day 3, but abolished it on day 4. The site of Act-D’s blockade of estrogen-induced LH release was investigated. Act-D treatment suppressed serum levels of LRF while it failed to modify either hypothalamic LRF content or noon levels of pituitary and serum LH on day 4. Act-D treatment consistently reduced the weight and the ribonucleic acid (RNA) content of the pituitary; a similar effect on RNA in the medial basal hypothalamus was not detected on day 4. On the other hand, electrochemical stimulation of the medial preoptic area of Act-D-treated rats on day 4 raised plasma LH significantly. However, the elevations at 45 min after stimulation were less in Act-D-treated than in control rats. Similarly, a significantly smaller response of LH release was observed at 15 min following intravenous injections of LRF in Act-D rats than in controls. These studies suggest that Act-D-sensitive steps exist at more than one site on the preopticotuberal pituitary axis involved in the stimulatory feedback action of estrogen on LH releas