To determine whether the plasma membrane is a primary site for progesterone (P4) action on the neural LHRH apparatus of hypothalamic tissues derived from ovariectomized, estradiol-primed (OVX + E2) immature rats, immobilized P4 was infused directly to these tissues using a superfusion technique. Two kinds of immobilized P4 with bovine serum albumin (BSA) conjugated at positions 3 or 11, or 11-deoxycorticosterone (DOC) immobilized at position 21 of the steroid molecule, respectively, were tested for structural specificity. Among the three immobilized steroids, only P4 with BSA conjugated at position 3 (P4-3-BSA) was effective in stimulating LHRH release in vitro. P4-3-BSA at 0.5 micrograms/ml, approximately 1.7 X 10(-7) M of P4, increased LHRH levels in the superfusates to about 2.5-fold those of pretreatment levels. In addition, no conversion of P4-3-BSA to free progesterone was detected. This observation demonstrated that the plasma membrane is a primary site for the stimulating effect of P4 on LHRH release from hypothalamic tissue in vitro.