This paper deals with the effect of temperature (t degrees), ions, prostaglandin (PGE2) and testosterone on the rate of release of LHRH from the superfused medial basal hypothalamus (MBH) of normal or castrated male rats in an effort to further validate the biological properties of this preparation. LHRH in the perfusate is nondetectable when the MBH is at 0 C, warming to 37 C produces a sharp rise in LHRH and then stabilization at a level at least threefold the one calculated for the 0 C experiment. Basal release rate of LHRH is t degrees and calcium dependent, as is the stimulatory action of potassium. Infusion of high K+ induces approximately a fivefold increase in the release rate of LHRH and stays at this high level for as long as the infusion lasts (50 min). LHRH stimulation by PGE2 (pulse) proceeds in the absence of external calcium, and the effect lasts longer than the potassium (pulse) action. Surprisingly, MBHs from long-ter castrated males (60-90 days) release less LHRH per minute than intact male MBHs during the steady state phase of LHRH secretion; testosterone propionate (TP, 200 microgram/100 g BW x 3 days) increases significantly the rate of release in castrate rats and is not related to the amount of LHRH assayed in the MBH. MBHs of untreated castrated rats or castrated rats treated with TP, show a reduced LHRH secretion in response to PGE2. It is concluded that this preparation can be used at advantage to study the rate of LHRH release in a variety of experimental paradigms.