1AbstractThe diameter of the pupil fluctuates in response to levels of ambient light and is regulated by the autonomic nervous system. Increasing light in one eye causes both pupils to constrict, implying the system must combine signals across the two eyes – a process of binocular integration occurring independently of visual cortex. Distinct classes of retinal photoreceptor are involved in controlling and maintaining pupil diameter, with cones and rods driving the initial constriction and intrinsically photosensitive retinal ganglion cells maintaining diameter over prolonged time periods. Here, we investigate binocular combination by targeting different photoreceptor pathways using the silent substitution method to modulate the input spectra. We find different patterns of binocular response in each pathway, and across the first and second harmonic frequencies. At the first harmonic, luminance and S-cone responses showed strong binocular facilitation, and weak interocular suppression. Melanopsin responses were invariant to the number of eyes stimulated. Notably, the L-M pathway involved binocular inhibition, whereby responses to binocular stimulation were weaker than for monocular stimulation. The second harmonic involved strong interocular suppression in all pathways, but with some evidence of binocular facilitation. Our results are consistent with a computational model of binocular signal combination (implemented in a Bayesian hierarchical framework), in which the weight of interocular suppression differs across pathways. We also find pathway differences in response phase, consistent with different lag times for phototransduction. This work demonstrates for the first time the algorithm governing binocular combination in the autonomic nervous system.