Using extracellular single-unit recording in brain slices, we have tested the effects of serotonin and vasoactive intestinal peptide (VIP) on the action of glutamate on hypothalamic suprachiasmatic neurons. We hypothesized that serotonin and VIP may interact with glutamate so that a modulation of the circadian rhythm entrainment to the light-dark may be possible. Given individually, glutamate excited 32% and inhibited 1% (n = 65), serotonin excited 8% and inhibited 27% (n = 73), and VIP excited 7% and inhibited 7% (n = 41) of suprachiasmatic neurons. When administered together, however, serotonin or VIP not only increased the percentage of neurons (up to 50%) that responded to glutamate, but also potentiated the neuronal responses to glutamate. These findings substantiated our hypothesis that both serotonin and VIP may modulate the action of glutamate in the suprachiasmatic nucleus.