Previous studies have suggested that the stimulatory effect of neuropeptide Y (NPY) on the release of luteinizing hormone (LH) in rats may be due to a central action of the peptide that promotes the release of LH-releasing hormone (LHRH) from the hypothalamus, and to an action in the pituitary gland, to potentiate the release of LH induced by LHRH. The objectives of the present experiments were to test 1) whether NPY stimulation of LHRH release requires extracellular Ca++, and 2) whether NPY can exert direct stimulatory effects on the release of LH from anterior pituitary cells. The in vitro release of LHRH from medial basal hypothalamic fragments induced by KCl depolarization (56 mM), but not the basal release, was blocked by omission of Ca++ and addition of 0.1 mM EGTA to the incubation medium and also by cobalt (1 mM). Depolarization-induced release of the peptide was unaffected by nifedipine, diltiazem, or lanthanum. However, the stimulation of LHRH release by NPY (1 microM) still occurred in Ca++ free/EGTA medium. In a second set of experiments, 10 min pulses of NPY (1-100 nM) alone were ineffective in stimulating the release of LH from dispersed, perifused anterior pituitary cells obtained from ovariectomized, untreated or ovariectomized, estrogen-treated rats, under conditions where pulses of LHRH (0.1-10 nM) were consistently effective. A brief increase in LH release was observed during a 30 min exposure to 100 nM NPY in estrogen-pretreated cells, but not from untreated cells, and the effect was not as marked as that produced by LHRH.(ABSTRACT TRUNCATED AT 250 WORDS)