Translocation of pigment granules in X. laevis melanophores activated by light (photodispersion) is mediated by a vitamin A-based opsin photoreceptor. This study examined the expression of mRNAs encoding classical rod and cone opsins, and the novel non-image forming opsin, melanopsin, by quantitative real-time PCR (qPCR). In addition, the functional role of melanopsin was examined by inhibiting melanopsin expression using siRNA and examining the effect on photodispersion of pigment. Total RNA was isolated from melanophores and X. laevis retina, reverse transcribed with MMLV-RT and random 10-mers and qPCR done using primers specific for red rod, green rod, red cone, violet cone opsins and melanopsin. Melanopsin specific siRNAs were designed and prepared with the Silencer™ construction kit (Ambion) and transfected by electroporation. Pigment granule dispersion was measured in melanophores (6-8000/well) in 96-well plates by measuring the change in absorbance at 630 nm, or the change in pigmented area using ImageJ (http://rsb.info.nih.gov/ij/) in individual cells. Specific, sensitive qPCR assays readily measured all opsins in X. laevis retina cDNA, but could detect only melanopsin mRNA in melanophore cDNA (4.9 x 10³ copies/10⁸ copies 28S rRNA) indicating that melanopsin is the opsin mediating photodispersion. To examine its role further, a pool of four melanopsin-specific siRNAs were transfected into melanophores. Melanopsin mRNA expression was dramatically reduced (~70%) 3 days after transfection, yet expression of mRNAs encoding other GPCRs known to trigger pigment dispersion (5-HT₇ receptor) and aggregation (melatonin_1c receptor) was unaltered, and STAT1 (indicative of activation of the interferon response pathway) was not up-regulated. Transfection of melanophores with a negative control siRNA did not reduce melanopsin mRNA. Western blotting using a specific melanopsin antibody showed that melanopsin mRNA knock-down was accompanied by a reduction in melanopsin protein expression. Irradiance-response curves to white light were compared in negative control transfected and melanopsin siRNA transfected melanophores. Melanopsin knock-down reduced maximal photodispersion by 50 ± 4% (n=6), while the irradiance-response curve after transfection of a negative control siRNA was identical to untransfected cells. The inhibition of dispersion in siRNA transfected melanophores was specific to the light stimulus, as treatment with α-melanocyte stimulating hormone (1 nM) induced substantial additional dispersion (82 ± 2% of maximum, n=6). Measurement of pigmented area on individual siRNA transfected melanophores confirmed that photodispersion was reduced. These experiments show that light-induced dispersion of pigment granules in X. laevis melanophores is mediated by melanopsin, an opsin found in the mammalian retinal ganglion cells that mediate non-image forming functions such as the pupil light reflex, circadian photoentrainment and light inhibition of the synthesis of the pineal gland hormone, melatonin.