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Timing of the mammalian circadian clock of the suprachiasmatic nucleus (SCN) is regulated by photic input from the retina. Retinorecipient units entrain rhythmicity of SCN pacemaker cells in part through their release of vasoactive intestinal polypeptide (VIP). The underlying nature of this process is conjectural, however, as in-vivo SCN VIP release has never been measured. Here, SCN microdialysis was used to investigate mechanisms regulating VIP. Hamsters under light-dark cycle of 14 : 10 exhibited a daily peak in synaptic VIP release near midday. Under constant darkness, this output was arrhythmic. Light and the glutamatergic agonist, N-methyl-D-aspartate, stimulated VIP release at night, whereas the serotonin (1A,7) agonist, (±)8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide, suppressed release at midday. Hence, SCN VIP activity is stimulated by photic input and inhibited by serotonin.