GnRH stimulates secretion of pituitary LH by increasing intracellular calcium. Increased calcium may result from activation of phospholipase-C, since there is an increase in inositol phosphates and diacylglycerol, and a redistribution of protein kinase-C (PKC) from cytosolic to a particulate cell fraction in GnRH-stimulated pituitary cultures. A GTP-binding protein (G-protein) may mediate GnRH actions, since GTP stimulates LH release in permeabilized gonadotropes and decreases receptor affinity for a GnRH analog. In the present study we have used sodium fluoride, an exogenous activator of G-proteins, to investigate the possibility of a G-protein link between GnRH receptor activation, phospholipase-C activity, and LH release. Treatment of primary pituitary cell cultures from immature female rats with sodium fluoride stimulated the release of 20% total cellular LH and increased inositol phosphate accumulation. Sodium fluoride-stimulated LH release was insensitive to cholera toxin and pertussis toxin. Sodium fluoride-stimulated LH release was additive with a maximally effective concentration of phorbol 12-myristate 13-acetate and was not inhibited by depletion of cellular PKC, suggesting that PKC does not mediate sodium fluoride effects. Treatment of cultures with 3 mM EGTA and 10 nM GnRH for 5 and 16 h reduced pituitary responsiveness to subsequent treatment with GnRH, but had no effect on sodium fluoride-stimulated LH release. Although the precise mechanism of sodium fluoride-stimulated LH release remains to be described, our results support a role for a G-protein in regulation of LH release by the releasing hormone.