In mammals, nonvisual responses to light have been shown to involve intrinsically photosensitive retinal ganglion cells (ipRGC) that express melanopsin and that are modulated by input from both rods and cones. Recent in vitro evidence suggests that melanopsin possesses dual photosensory and photoisomerase functions, previously thought to be a unique feature of invertebrate rhabdomeric photopigments. In cultured cells that normally do not respond to light, heterologous expression of mammalian melanopsin confers light sensitivity that can be restored by prior stimulation with appropriate wavelengths. Using three different physiological and behavioral assays, we show that this in vitro property translates to in vivo, melanopsin-dependent nonvisual responses. We find that prestimulation with long-wavelength light not only restores but enhances single-unit responses of SCN neurons to 480-nm light, whereas the long-wavelength stimulus alone fails to elicit any response. Recordings in Opn4-/- mice confirm that melanopsin provides the main photosensory input to the SCN, and furthermore, demonstrate that melanopsin is required for response enhancement, because this capacity is abolished in the knockout mouse. The efficiency of the light-enhancement effect depends on wavelength, irradiance, and duration. Prior long-wavelength light exposure also enhances short-wavelength-induced phase shifts of locomotor activity and pupillary constriction, consistent with the expression of a photoisomerase-like function in nonvisual responses to light.