Regulators of G protein signaling (RGS) are proteins that bind specifically to activated Galpha subunits of heterotrimeric G proteins to terminate signaling by both Galpha and Gbetagamma subunits. Signal-induced RGS redistribution may affect their activity in G protein-mediated signaling. We have previously shown that melatonin and the cell permeable cGMP analog 8-bromo cGMP, which lead to protein kinase C (PKC) activation, enhanced cytoplasmic distribution of RGS10 and RGS2 in prostate carcinoma PC3-AR cells. In the present study, we transfected PC3-AR cells with myc-tagged Galphai/Galphaq specific RGS proteins RGS2, RGS4 and RGS10 and examined the effects of melatonin, 8-bromo cGMP and PKC inhibitors on their nuclear-cytoplasmic partitioning. RGS10 and RGS2 were predominantly localized in the nucleus and perinuclear regions whereas RGS4 was mostly cytoplasmic in the PC3-AR cells. Melatonin and the cell permeable cGMP analog 8-bromo cGMP, previously found to activate PKCalpha in the PC3-AR cells, enhanced cytoplasmic localization of RGS10 and RGS2 but induced nuclear accumulation of RGS4. The isozyme specific PKC inhibitor GO6976 (PKCalpha and PKCbeta1) but not hispidin (PKCbeta) negated the effects of melatonin on RGS10, RGS2 and RGS4 localization. These findings indicate that PKCalpha, a downstream effector of the melatonin receptor, differentially affects nuclear/cytoplasmic localization of both Galphai and Galphaq specific RGS proteins. These observations provide further insight into melatonin's ability to fine-tune multiple membrane G-proteins signaling in cells.