The suprachiasmatic nucleus (SCN) receives glutamatergic afferents from the retina and serotonergic afferents from the midbrain, and serotonin (5-HT) can modify the response of the SCN circadian oscillator to light. 5-HT1B receptor-mediated presynaptic inhibition has been proposed as one mechanism by which 5-HT modifies retinal input to the SCN (Pickard et al., 1996). This hypothesis was tested by examining the subcellular localization of 5-HT1B receptors in the mouse SCN using electron microscopic immunocytochemical analysis with 5-HT1B receptor antibodies and whole-cell patch-clamp recordings from SCN neurons in hamster hypothalamic slices. 5-HT1B receptor immunostaining was observed associated with the plasma membrane of retinal terminals in the SCN. 1-[3-(Trifluoromethyl)phenyl]-piperazine HCl (TFMPP), a 5-HT1B receptor agonist, reduced in a dose-related manner the amplitude of glutamatergic EPSCs evoked by stimulating selectively the optic nerve. Selective 5-HT1A or 5-HT7 receptor antagonists did not block this effect. Moreover, in cells demonstrating an evoked EPSC in response to optic nerve stimulation, TFMPP had no effect on the amplitude of inward currents generated by local application of glutamate. The effect of TFMPP on light-induced phase shifts was also examined using 5-HT1B receptor knock-out mice. TFMPP inhibited behavioral responses to light in wild-type mice but was ineffective in inhibiting light-induced phase shifts in 5-HT1B receptor knock-out mice. The results indicate that 5-HT can reduce retinal input to the circadian system by acting at presynaptic 5-HT1B receptors located on retinal axons in the SCN.