Neuroactive peptides and the intracellular calcium concentration ([Ca2+]i) play important roles in light-induced modulation of gene expression in the suprachiasmatic nucleus (SCN) neurons that ultimately control behavioral rhythms. Vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) are expressed rhythmically within populations of SCN neurons. Pituitary adenylate cyclase-activating peptide (PACAP) is released from retinohypothalamic tract (RHT) terminals synapsing on SCN neurons. Nociceptin/orphanin FQ (OFQ) receptors are functionally expressed in the SCN. We examined the role of several neuropeptides on Ca2+ signaling, simultaneously imaging multiple neurons within the SCN neural network. VIP reduced the [Ca2+]i in populations of SCN neurons during the day, but had little effect at night. Stimulation of the RHT at frequencies that simulate light input signaling evoked transient [Ca2+]i elevations that were not altered by VIP. AVP elevated the [Ca2+]i during both the day and night, PACAP produced variable responses, and OFQ induced a reduction in the [Ca2+]i similar to VIP. During the day, VIP lowered the [Ca2+]i to near nighttime levels, while AVP elevated [Ca2+]i during both the day and night, suggesting that the VIP effects on [Ca2+]i were dependent, and the AVP effects independent of the action potential firing activity state of the neuron. We hypothesize that VIP and AVP regulate, at least in part, Ca2+ homeostasis in SCN neurons and may be a major point of regulation for SCN neuronal synchronization.