Mammals contain 1 melanopsin (Opn4) gene that is expressed in a subset of retinal ganglion cells to serve as a photopigment involved in non-image-forming vision such as photoentrainment of circadian rhythms. In contrast, most nonmammalian vertebrates possess multiple melanopsins that are distributed in various types of retinal cells; however, their functions remain unclear. We previously found that the lamprey has only 1 type of mammalian-like melanopsin gene, which is similar to that observed in mammals. Here we investigated the molecular properties and localization of melanopsin in the lamprey and other cyclostome hagfish retinas, which contribute to visual functions including image-forming vision and mainly to non-image-forming vision, respectively. We isolated 1 type of mammalian-like melanopsin cDNA from the eyes of each species. We showed that the recombinant lamprey melanopsin was a blue light-sensitive pigment and that both the lamprey and hagfish melanopsins caused light-dependent increases in calcium ion concentration in cultured cells in a manner that was similar to that observed for mammalian melanopsins. We observed that melanopsin was distributed in several types of retinal cells, including horizontal cells and ganglion cells, in the lamprey retina, despite the existence of only 1 melanopsin gene in the lamprey. In contrast, melanopsin was almost specifically distributed to retinal ganglion cells in the hagfish retina. Furthermore, we found that the melanopsin-expressing horizontal cells connected to the rhodopsin-containing short photoreceptor cells in the lamprey. Taken together, our findings suggest that in cyclostomes, the global distribution of melanopsin in retinal cells might not be related to the melanopsin gene number but to the extent of retinal contribution to visual function.