The goal of the present study was to investigate a possible role for regulators of G protein-signaling (RGS) proteins in opioid receptor (OR) desensitization using cultured Xenopus laevis dermal melanophores. Morphine-induced pigment aggregation in a melanophore cell line stably expressing the murine mu OR (muOR) was quantified over time. Responses of the muOR (a G(i)-linked receptor) exhibited a time-dependent desensitization, which varied with the concentration of morphine used. In contrast, much less desensitization was observed in response to melatonin, effects mediated through the cells' endogenous melatonin receptor (which is also G(i)-linked). To further study OR desensitization, melanophores lacking a muOR were transiently transfected with plasmids encoding the muOR alone or in combination with plasmids encoding one of several RGS subtypes (RGS1, RGS2, RGS3, or RGS4). Overexpression of RGS2, but not the other RGS subtypes, produced a rightward shift in the morphine concentration-response curve. RGS protein overexpression also decreased the magnitude of morphine-induced responses. Finally, the effect of a mutant form of Galpha(i1), which is insensitive to RGS action, was investigated with respect to its ability to alter the response of the muOR to morphine. Expression of the mutant Galpha(i1) prolonged morphine-induced pigment aggregation and produced leftward shifts in concentration-response curves, compared with expression of wild-type Galpha(i1). These results demonstrate that specific RGS proteins can dampen signals initiated by agonist activation of the muOR, and support a possible role for RGS proteins in OR desensitization.