The study examined the effect of estrogen priming on progesterone (P4)-induced LHRH release, the tissue site of action of P4, and the effect of 5 alpha-dihydroxyprogesterone (5 alpha-DHP) on LHRH release from hypothalamic fragments superfused in vitro. Immature female rats were ovariectomized (OVX) and, at 28 days of age, Silastic capsules containing estradiol (E2) were implanted. Two days later, animals were killed and hypothalamic fragments were removed and transferred to superfusion chambers. The hypothalamic units received P4 or 5 alpha-DHP delivered in an intermittent mode (10-min on, 20-min off). LHRH was determined in perfusates by RIA. After the input characteristics of different infusion modes (single pulses, intermittent, and continuous) of P4 infused into superfusion chambers were assessed, an intermittent infusion mode (10-min on, 20-min off) was selected for further examinations. In the mediobasal hypothalamic-anterior hypothalamic-preoptic area (MBH-AHA-POA) tissue preparations, we observed: 1) an infusion of 5 alpha-DHP was ineffective in stimulating LHRH release; 2) the release pattern of LHRH in response to three different P4 doses (10, 20, and 50 ng/ml) was similar in terms of percent changes (202% to 219% over control values); and 3) E2 priming was absolutely required for P4-stimulated LHRH release, and this requirement appeared to be dose dependent. Upon an examination of three hypothalamic tissue boundaries [the MBH, the POA-suprachiasmatic nuclei (POA-SCN), and the median eminence (ME)] to better delineate the in vitro site of action of P4 on LHRH release, it was demonstrated that the MBH responded upon P4 infusion, whereas the POA-SCN was unable to do so. The ME also responded upon P4 infusion, and LHRH release followed closely the pulsatile administration of P4 since upon each challenge of the steroid at the concentration of 10 or 20 ng/ml, a significant rise in LHRH release occurred. However, the temporal patterns of LHRH release from the ME appears to be different from those obtained from the MBH as well as the MBH-AHA-POA. These observations demonstrate that an intermittent infusion of P4, but not 5 alpha-DHP, is effective in activating the neural LHRH apparatus. Estrogen is an obligatory requirement for this P4-stimulated LHRH release, and the neural site of action of P4 resides within the MBH. However, this steroid also can act directly upon the ME nerve terminals to release LHRH.