The retinohypothalamic tract, a monosynaptic retinal projection to the suprachiasmatic nucleus (SCN), is the path by which light entrains the circadian system to the external photoperiod. Serotonergic neurons in the mesencephalic median raphe nucleus (MnR) also give rise to a major SCN afferent projection. The present study was designed to determine the extent to which MnR serotonergic projections regulate sensitivity of the circadian rhythm system to light. Serotonergic neurons in the MnR were destroyed by the direct application of the neurotoxin, 5,7-dihydroxytryptamine. Animals in constant darkness were given 5-min white light pulses at circadian time 19. Light intensity varied from 0.0011 to 70 microW/cm2. Assessment of rhythm phase response to light by lesioned and control animals revealed that animals lacking the MnR serotonergic projection are considerably more sensitive to light at high irradiances. The results are consistent with behavioral and physiological evidence implicating serotonin as an inhibitory modulator of the effects of light on circadian rhythmicity.