In addition to rods and cones, the human retina contains melanopsin which has been identified recently in the body and dendrites of a few ganglion cells. The intrinsically photosensitive retinal ganglion cells (ipRGCs) are good candidates for controlling the tonic pupil aperture but their spectral sensitivity is close to those of rods and S-cones which are other candidates. Our study aims at identifying the stimulus for the pupil response when the luminance is constant and the spectrum of the light changes. A light booth was equipped with five types of coloured light emitting diodes (LEDs): Blue, Cyan, Green, Orange and Red. The intensity of each LED type could be adjusted to control the light spectrum. Illumination pairs were prepared ensuring the exclusive variation of excitation of one receptor type and silent substitution for others. Because the range of the possible controlled changes of excitation was narrow, we also prepared illumination pairs ensuring silent substitution for luminance rather than for L-cones and M-cones independently. Photographs of the observer's eyes were taken following one minute of adaptation to each illumination and the ratio of pupil to iris diameter was measured. No differential pupillary response was observed with a variation of rod, melanopsin or S-cone excitation alone. A differential pupillary response could only be obtained with a variation of the melanopsin stimulus of sufficient high contrast with or without a concurrent variation of rods.