Light is the major synchronizer of circadian rhythms to the 24-h solar day. The intrinsically photosensitive retinal ganglion cells (ipRGCs) play a central role in circadian regulation but cones also provide, albeit indirectly, input to these cells. In humans, spectrally opponent blue versus yellow (b-y) bipolar cells lying distal to the ganglion cell layer were hypothesized to provide direct input to the ipRGCs and therefore, the circadian system should exhibit subadditivity to some types of polychromatic light. Ten subjects participated in a within-subjects 3-night protocol. Three experimental conditions were employed that provided the same total irradiance at both eyes: (1) one unit of blue light (lambda(max)=450 nm, 0.077 W/m(2)) to the left eye plus one unit of green light (lambda(max)=525 nm, 0.211 W/m(2)) to the right eye, (2) one unit of blue light to the right eye plus one unit of green light to the left eye, and (3) 1/2 unit of blue light plus 1/2 unit of green light to both eyes. The first two conditions did not differ significantly in melatonin suppression while the third condition had significantly less melatonin suppression than conditions 1 and 2. Furthermore, the magnitudes of suppression were well predicted by a previously published model of circadian phototransduction incorporating spectral opponency. As was previously demonstrated, these results show that the human circadian system exhibits a subadditive response to certain polychromatic light spectra. This study demonstrates for the first time that subadditivity is due to spectrally opponent (color) retinal neurons.