Glutamate is considered to be the primary neurotransmitter in the retinohypothalamic tract (RHT), which delivers photic information from the retina to the suprachiasmatic nucleus (SCN), the locus of the mammalian circadian pacemaker. However, substance P (SP) also has been suggested to play a role in retinohypothalamic transmission. In this study, we sought evidence that SP from the RHT contributes to photic resetting of the circadian pacemaker and further explored the possible interaction of SP with glutamate in this process. In rat hypothalamic slices cut parasagittally, electrical stimulation of the optic nerve in early and late subjective night produced a phase delay (2.4 +/- 0.5 hr; mean +/- SEM) and advance (2.6 +/- 0.3 hr) of the circadian rhythm of SCN neuronal firing activity, respectively. The SP antagonist L-703,606 (10 microm) applied to the slices during the nerve stimulation completely blocked the phase shifts. Likewise, a cocktail of NMDA (2-amino-5-phosphonopentanoic acid, 50 microm) and non-NMDA (6,7-dinitroquinoxaline-2,3-dione, 10 microm) antagonists completely blocked the shifts. Exogenous application of SP (1 microm) or glutamate (100 microm) to the slices in early subjective night produced a phase delay ( approximately 3 hr) of the circadian firing activity rhythm of SCN neurons. Coapplication of the NMDA and non-NMDA antagonist cocktail (as well as L-703,606) resulted in a complete blockade of the SP-induced phase delay, whereas L-703,606 (10 microm) had no effect on the glutamate-induced delay. These results suggest that SP, as well as glutamate, has a critical role in photic resetting. Furthermore, the results suggest that the two agonists act in series, SP working upstream of glutamate.