The suprachiasmatic nucleus (SCN) is the central circadian clock, governing physiological processes through its output to the subparaventricular zone (SPZ). We investigated functional synaptic connectivity between the SCN and the SPZ neurons in mouse brain slices and we studied the effects of peptides expressed in the SCN on the SPZ neurons. Photostimulation of SCN neuromedin-S (NMS) neurons or the vasoactive intestinal peptide (VIP) neurons induced opto-evoked inhibitory post-synaptic currents (oIPSCs) in 60% and 50% of recorded SPZ neurons, respectively. These responses were mediated by GABAA signaling, and they were maintained in tetrodotoxin (TTX) indicating monosynaptic connectivity. Using calcium imaging recordings, we found that VIP, NMS, and arginine vasopressin (AVP), three peptides co-expressed in subsets of NMS neurons, directly activated dorsal (dSPZ) and ventral SPZ (vSPZ) neurons. All recordings were conducted in TTX indicating the direct effects of these three peptides. We found that VIP excited 29%, NMS 6% and AVP 38% of the vSPZ neurons and 42%, 61% and 55% of the dSPZ neurons. BAY 55-9837, a VPAC2 receptor agonist activated SPZ neurons in both vSPZ and dSPZ. The net effect of the co-release of GABA, an inhibitory signal, and any of the three peptides (NMS, VIP, or AVP) that we found activate the SPZ neurons remains unclear. However, considering that in nocturnal animals the activity of the SCN neurons is in antiphase with the activity of the SPZ neurons, it is likely that GABA signaling produces a more prominent influence on SPZ neuronal activity, than the peptides do.