Negative masking of locomotor activity by light in nocturnal rodents is mediated by a non-image-forming irradiance-detection system in the retina. Structures receiving input from this system potentially contribute to the masking response. The suprachiasmatic nucleus (SCN) regulates locomotor activity and receives dense innervation from the irradiance-detection system via the retinohypothalamic tract, but its role in masking is unclear. We studied masking in adult Syrian hamsters (Mesocricetus auratus) with electrolytic lesions directed at the SCN. Hamsters were exposed to a 3.5:3.5 ultradian light/dark cycle and their wheel-running activity was monitored. Intact hamsters showed robust masking, expressing less than 20% of their activity in the light even though light and dark occurred equally during their active times. In contrast, hamsters with lesions showed, on average, as much activity in the light as in the dark. Tracing of retinal projections using cholera toxin beta subunit showed that the lesions damaged retinal projections to the SCN and to the adjacent subparaventricular zone. Retinal innervation outside the hypothalamus was not obviously affected by the lesions. Our results indicate that retinohypothalamic projections, and the targets of these projections, to the SCN and/or adjacent hypothalamic areas play an important role in masking.