Neuropeptide Y (NPY) has been shown to be capable of both the enhancement and suppression of gonadotropin secretion from pituitary cells. In order to elucidate the underlying cellular mechanisms which might account for these actions, we have examined the effects of NPY on gonadotropin secretion stimulated by either cell depolarization or by GnRH from primary cultures of rat pituitary cells. In one set of experiments, we measured single-cell [Ca2+]i using the Ca2(+)-sensitive intracellular fluorescent indicator Fura-2, in gonadotropes which had been identified using a reverse hemolytic plaque assay employing an antiserum to LH. In another group of investigations, we measured FSH and LH secretion in response to depolarization or stimulation with GnRH, and examined the influence of NPY on these patterns of secretion. NPY was active in inhibiting the amplitude of the [Ca2+]i signal induced by depolarization with 20 mM K+, as well as in substantially blocking the secondary plateau phase of the GnRH-induced elevation of [Ca2+]i. However, the peak [Ca2+]i transients occurring in response to either depolarization with 50 mM K+ or the initial phase of the GnRH-induced response, were not sensitive to blockade by NPY. Moreover, treatment of the cells for 24 h with pertussis toxin prevented the NPY-mediated inhibition of the GnRH-stimulated [Ca2+]i plateau. Cell depolarization by 50 mM K+ induced 3-fold increases in FSH and LH release over 2-h incubations. GnRH (100 nM) elicited a 9-fold increase in FSH and a 14-fold stimulation of LH over the same time period. NPY had insignificant effects upon depolarization-induced hormone release, but at 1 microM partially suppressed LH release elicited by 100 nM GnRH over 2 h. We conclude that NPY is capable of inhibiting [Ca2+]i signals in gonadotropes that are stimulated by GnRH, and that these effects are mediated through activation of a pertussis toxin-sensitive G-protein. These effects on [Ca2+]i may underly the inhibitory effects of NPY on gonadotropin secretion.