In mammals, melanopsin is exclusively expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs), which play an important role in circadian photoentrainment and other nonimage-forming functions. These ipRGCs reside in the inner retina, far removed from the pigment epithelium, which synthesizes the 11-cis retinal chromophore used by rod and cone photoreceptors to regenerate opsin for light detection. There has been considerable interest in the identification of the melanopsin chromophore and in understanding the process of photopigment regeneration in photoreceptors that are not in proximity to the classical visual cycle. We have devised an immuno-magnetic purification protocol that allows melanopsin-expressing retinal ganglion cells to be isolated and collected from multiple mouse retinas. Using this technique, we have demonstrated that native melanopsin in vivo exclusively binds 11-cis retinal in the dark and that illumination causes isomerization to the all-trans isoform. Furthermore, spectral analysis of the melanopsin photoproduct shows the formation of a protonated metarhodopsin with a maximum absorbance between 520 and 540 nm. These results indicate that even if melanopsin functions as a bistable photopigment with photo-regenerative activity native melanopsin must also use some other light-independent retinoid regeneration mechanism to return to the dark state, where all of the retinal is observed to be in the 11-cis form.