The phase of the mammalian circadian pacemaker, located in the suprachiasmatic nucleus (SCN), is modulated by a variety of stimuli, most notably the environmental light cycle. Light information is perceived by the circadian pacemaker through glutamate that is released from retinal ganglion cell terminals in the SCN. Other prominent modulatory inputs to the SCN include a serotonergic projection from the raphe nuclei and a neuropeptide Y (NPY) input from the intergeniculate leaflet. Light and glutamate phase-shift the SCN pacemaker at night, whereas serotonin (5-HT) and NPY primarily phase-shift the pacemaker during the day. In addition to directly phase-shifting the circadian pacemaker, SCN inputs have been shown to modulate the actions of one another. For example, 5-HT can inhibit the phase-shifting effects of light or glutamate applied to the SCN at night, and NPY and glutamate inhibit phase shifts of one another. In this study, we explored the possibility that glutamate can modulate serotonergic phase shifts during the day. For these experiments, we applied various combinations of 5-HT agonists, glutamate agonists, and electrical stimulation of the optic chiasm to SCN brain slices to determine the effect of these treatments on the rhythm of spontaneous neuronal activity generated by the SCN circadian pacemaker. We found that glutamate agonists and optic chiasm stimulation inhibit serotonergic phase advances and that this inhibition involves both AMPA and NMDA receptors. This inhibition by glutamate may be indirect, because it is blocked by both tetrodotoxin and the GABA(A) antagonist, bicuculline.