Dopamine is the primary inhibitory regulator of lactotroph proliferation and PRL secretion in vivo, acting via dopamine D2 receptors (short D2S and long D2L forms). In GH₄C₁ pituitary cells transfected with D2S or D2L receptor cDNA, dopamine inhibits PRL secretion and DNA synthesis. These actions were blocked by pertussis toxin, implicating G_i/G_o proteins. To address roles of specific G_i/G_o proteins in these actions a series of GH₄C₁ cell lines specifically depleted of individual G subunits was examined. D2S-mediated inhibition of BayK8644-stimulated PRL secretion was primarily dependent on G_o over G_i, as observed for BayK8644-induced calcium influx. By contrast, inhibitory coupling of the D2S receptor to TRH-induced PRL secretion was partially impaired by depletion of any single G protein, but especially G_i3. Inhibitory coupling of D2L receptors to PRL secretion required G_o, but not G_i2, muscarinic receptor coupling was resistant to depletion of any G_i/G_o protein, whereas the 5-HT1A and somatostatin receptors required G_i2 or G_i3 for coupling. The various receptors also demonstrated distinct G protein requirements for inhibition of DNA synthesis: depletion of any G_i/G_o subunit completely uncoupled the D2S receptor, the D2L receptor was uncoupled by depletion of G_o or G_i2, and muscarinic and somatostatin receptors were resistant to depletion of G_i2 only. These results demonstrate distinct receptor-G protein preferences for inhibition of TRH-induced PRL secretion and DNA synthesis.