The excitability of gonadotropin-releasing hormone (GnRH) neurons is essential for episodic neuropeptide release, but the mechanism by which electrical activity controls GnRH secretion is not well characterized. The role of phospholipase D (PLD) in mediating the activity-dependent secretory pathway was investigated in immortalized GT1 neurons, which both secrete GnRH and express GnRH receptors. Activation of these Ca2+-mobilizing receptors was associated with transient hyperpolarization of GT1 cells, followed by sustained firing of action potentials. This was accompanied by an increase in PLD activity, as indicated by elevated phosphatidylethanol (PEt) production. GnRH-induced PEt production was reduced by inhibition of phospholipase C-dependent phosphoinositide hydrolysis by U73122 and neomycin, suggesting that signaling from phospholipase C led to activation of PLD. The intermediate role of protein kinase C (PKC) in this process was indicated by the ability of phorbol 12-myristate 13-acetate to induce time- and dose-dependent increases in PEt and diacylglycerol, but not inositol trisphosphate, and by reduction of GnRH-induced PEt accumulation in PKC-depleted cells. Consistent with the role of action potential-driven Ca2+ entry in this process, agonist-induced PLD activity was also reduced by nifedipine and low extracellular Ca2+. Inhibition of the PLD pathway by ethanol and propranolol reduced diacylglycerol production and caused a concomitant fall in GnRH release. These data indicate that voltage-gated Ca2+ entry and PKC act in an independent but cooperative manner to regulate PLD activity, which contributes to the secretory response in GT1 cells. Thus, the electrical activity of the GnRH-secreting neuron participates in the functional coupling between GnRH receptors and PLD pathway.