Melanopsin, expressed in a subset of intrinsically photosensitive ganglion cells that project to the suprachiasmatic nucleus (SCN), is involved in the photic entrainment of circadian rhythms and other non-image-forming functions (pupil light reflex, masking, acute heart rate response, and alertness). Melanopsin has recently been shown to be a "bireactive" photopigment that functions as a photosensory opsin using 11-cis retinaldehyde as a chromophore and has intrinsic photoisomerase activity. Melanopsin is widely distributed in the retina of vertebrates and, depending on the species, is expressed in ganglion, amacrine, horizontal, and photoreceptor cells. The present study was conducted to determine the distribution of this opsin in the human retina. Human donor eyes were obtained from donors and fixed shortly after death. Immunohistochemistry was used to determine melanopsin expression in the retinas of three donors. The possible coexpression of this photopigment with other opsins was studied by double-labeling immunocytochemistry and confocal analysis. In addition to the expected labeling in ganglion cells of the inner retinal layers, an unexpected finding showed melanopsin-immunopositive label in the outer segments of cones that did not coexpress other known opsins. These melanopsin-expressing cones are extremely sparse (5-25 cones/mm2; 0.1%-0.5% of the entire cone population) and are located in the peripheral retina. The presence of melanopsin in human cones suggests image and non-image-forming roles in visual responses at both the cone input and ganglion cell output stages and their involvement in a broad spectrum of irradiance detection functions in the visual system.