The suprachiasmatic nucleus (SCN), a circadian pacemaker in the mammalian brain, receives photic information directly from the retina via the retinohypothalamic tract (RHT). Although the neurotransmitters of the RHT have not yet been identified, it is known that glutamate (Glu) and substance P (SP) are present in RHT axons. We report the responses of spontaneously firing SCN neurons to Glu and SP examined in isolated brain slices. 43% of the neurons show an excitatory response (an increase in firing rate) and 11% an inhibitory response to bath-applied SP at a concentration of 10(-7) M. Glu evokes excitatory responses from SCN neurons in a dose-dependent manner (10(-6)-10(-4) M). No day-night difference is observed in the response of SCN neurons either to 10(-7) M SP or to 10(-4) M Glu. Bath-applied SP has additive effects on Glu-evoked responses and pressure-ejected SP at a concentration of 0.8 mM strongly potentiates Glu responses. These results are consistent with the view that Glu and/or SP function as neurotransmitters, or modulators, in the RHT and suggest that cellular processes downstream of the activation of SP or Glu receptors mediate time-dependent phase responses of SCN neurons.