Serotonergic innervation is believed to inhibit the effects of light on the mammalian circadian timing system. Two anatomical components of this system, the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL), receive serotonergic input from midbrain raphe nuclei. The present studies use retrograde and anterograde tracing as well as neurotoxic lesion techniques to demonstrate that serotonergic cells in the median raphe nucleus (MR) project to the SCN and that serotonergic cells in the dorsal raphe nucleus (DR) project to the IGL. Neurotoxic lesions were also used to investigate the effects of selective serotonin (5-HT) neuron loss in the MR or DR on circadian rhythm parameters of animals entrained to a light/dark cycle or housed in constant light. 5-HT depletion in the MR, but not in the DR, induces an advance in onset, a delay in offset, and a longer duration of the nocturnal running-wheel activity phase. Circadian rhythm disruption in constant light is also more frequent in hamsters with MR lesions. A second experiment was designed to investigate the relationship between lesion location, 5-HT-immunoreactive (5-HT-IR) fiber loss, and behavioral changes. Destruction of 5-HT neurons in the MR causes 5-HT- IR fiber loss in the SCN, which may account for the observed changes in circadian parameters. DR lesions result in 5-HT-IR fiber depletion of the IGL, with no associated changes in the entrained rhythm. The anatomical and behavioral results support the view that a 5-HT projection from the MR mediates 5-HT effects on circadian rhythm regulation in hamsters.