Dopamine D2 receptors signal through the pertussis toxin (PTX)-sensitive G_i/o and PTX-insensitive G_z proteins, as well as through a G protein-independent, -arrestin/GSK3-depen-dent pathway. Activation of these receptors in pituitary lactotrophs leads to inhibition of prolactin (PRL) release. It has been suggested that this inhibition occurs through the G_i/o- protein-mediated inhibition of cAMP production and/or G_i/o- dimer-mediated activation of inward rectifier K⁺ channels and inhibition of voltage-gated Ca²⁺ channels. Here we show that the dopamine agonist-induced inhibition of spontaneous Ca²⁺ influx and release of pre-stored PRL was preserved when cAMP levels were elevated by forskolin treatment. We further observed that dopamine agonists inhibited both spontaneous and depolarization-induced Ca²⁺ influx in untreated but not in PTX-treated cells. This inhibition was also observed in cells with blocked inward rectifier K⁺ channels, suggesting that dopamine effect on voltage-gated Ca²⁺ channel gating is sufficient to inhibit spontaneous Ca²⁺ influx. However, agonist-induced inhibition of PRL release was only partially relieved in PTX-treated cells, indicating that dopamine receptors also inhibit exocytosis downstream of voltage-gated Ca²⁺ influx. The PTX-insensitive step in agonist-induced inhibition of PRL release was not affected by the addition of wortmannin, an inhibitor of PI3-kinase, and lithium, an inhibitor of GSK-3, but was attenuated in the presence of phorbol ester PMA, which inhibits G_z signaling pathway in a protein kinase C-dependent manner. Thus, dopamine inhibits basal PRL release by blocking voltage-gated Ca²⁺ influx through the PTX-sensitive-signaling pathway and by desensitizing Ca²⁺-secretion coupling through the PTX-insensitive and protein kinase C-sensitive signaling pathway.