Neurons in the mammalian suprachiasmatic nuclei (SCN) generate daily rhythms in physiology and behavior, but it is unclear how they maintain and synchronize these rhythms in vivo. We hypothesized that parallel signaling pathways in the SCN are required to synchronize rhythms in these neurons for coherent output. We recorded firing and clock-gene expression patterns while blocking candidate signaling pathways for at least 8 days. GABA_A and GABA_B antagonism increased circadian peak firing rates and rhythm precision of cultured SCN neurons, but G_i/o did not impair synchrony or rhythmicity. In contrast, inhibiting G_i/o with pertussis toxin abolished rhythms in most neurons and desynchronized the population, phenocopying the loss of vasoactive intestinal polypeptide (VIP). Daily VIP receptor agonist treatment restored synchrony and rhythmicity to VIP^−/− SCN cultures during continuous GABA receptor antagonism but not during G_i/o blockade. Pertussis toxin did not affect circadian cycling of the liver, suggesting that G_i/o plays a specialized role in maintaining SCN rhythmicity. We conclude that endogenous GABA controls the amplitude of SCN neuronal rhythms by reducing daytime firing, whereas G_i/o signaling suppresses nighttime firing, and it is necessary for synchrony among SCN neurons. We propose that G_i/o, not GABA activity, converges with VIP signaling to maintain and coordinate rhythms among SCN neurons.