Intracellular free Ca²⁺ regulates diverse cellular processes, including membrane potential, neurotransmitter release, and gene expression. To examine the cellular mechanisms underlying the generation of circadian rhythms, nucleus-targeted and untargeted cDNAs encoding a Ca²⁺-sensitive fluorescent protein (cameleon) were transfected into organotypic cultures of mouse suprachiasmatic nucleus (SCN), the primary circadian pacemaker. Circadian rhythms in cytosolic but not nuclear Ca²⁺ concentration were observed in SCN neurons. The cytosolic Ca²⁺ rhythm period matched the circadian multiple-unit-activity (MUA)-rhythm period monitored using a multiple-electrode array, with a mean advance in phase of 4 hr. Tetrodotoxin blocked MUA, but not Ca²⁺ rhythms, while ryanodine damped both Ca²⁺ and MUA rhythms. These results demonstrate cytosolic Ca²⁺ rhythms regulated by the release of Ca²⁺ from ryanodine-sensitive stores in SCN neurons.