The formation of G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) heteromers enables signaling diversification and holds great promise for improved drug selectivity. Most studies of these oligomerization events have been conducted in heterologous expression systems, and in vivo validation is lacking in most cases, thus questioning the physiological significance of GPCR heteromerization. The melatonin receptors MT1 and MT2 exist as homomers and heteromers when expressed in cultured cells. We showed that melatonin MT1/MT2 heteromers mediated the effect of melatonin on the light sensitivity of rod photoreceptors in mice. This effect of melatonin involved activation of the heteromer-specific phospholipase C and protein kinase C (PLC/PKC) pathway and was abolished in MT1(-/-) or MT2(-/-) mice, as well as in mice overexpressing a nonfunctional MT2 mutant that interfered with the formation of functional MT1/MT2 heteromers in photoreceptor cells. Not only does this study establish an essential role of melatonin receptor heteromers in retinal function, it also provides in vivo support for the physiological importance of GPCR heteromerization. Thus, the MT1/MT2 heteromer complex may provide a specific pharmacological target to improve photoreceptor function.