Information about environmental lighting conditions is conveyed to the suprachiasmatic nucleus (SCN), at least in part, via a glutamatergic fiber pathway originating in the retina, known as the retinohypothalamic tract (RHT). Previous work indicates that serotonin (5HT) can inhibit this pathway, although the underlying mechanisms are unknown. The authors became interested in the possibility that 5HT can inhibit the glutamatergic regulation of Ca2+ in SCN neurons and, by this mechanism, modulate light-induced phase shifts of the circadian system. To start to examine this hypothesis, optical techniques were used to measure Ca2+ levels in SCN cells in a brain slice preparation. First, it was found that 5HT produced a reversible and significant inhibition of Ca2+ transients evoked by synaptic stimulation. Next, it was found that 5HT did not alter the magnitude or duration of Ca2+ transients evoked by the bath application of glutamate or N-methyl-D-aspartate acid (NMDA) in the presence of tetrodotoxin (TTX). The authors feel that the simplest explanation for these results is that 5HT can act presynaptically at the RHT/SCN synaptic connection to inhibit the release of glutamate. The demonstration that 5HT can have a dramatic modulatory action on synaptic-evoked Ca2+ transients measured in SCN neurons adds support to the notion that the serotonergic innervation of the SCN may function to regulate environmental input to the circadian system. In addition, it was found that the administration of higher concentrations of 5HT can increase Ca2+ in at least a subpopulation of SCN neurons. This effect of 5HT was concentration dependent and blocked by a broad-spectrum 5HT antagonist (metergoline). In addition, both TTX and the gamma-amino-N-butyric acid (GABA) receptor blocker bicuculline inhibited the 5HT-induced Ca2+ transients. Therefore, the interpretation of this data is that 5HT can act within the SCN to alter GABAergic activity and, by this mechanism, cause changes in intracellular Ca2+. It is also suggested that this 5HT-induced Ca2+ increase might play a role in 5HT-induced phase shifts of the SCN circadian oscillator.