Generation of mammalian circadian rhythms involves molecular transcriptional and translational feedback loops. It is not clear how membrane events interact with the intracellular molecular clock or whether membrane activities are involved in the actual generation of the circadian rhythm. We examined the role of membrane potential and calcium (Ca²⁺) influx in the expression of the circadian rhythm of the clock gene Period 1 (Per1) within the rat suprachiasmatic nucleus (SCN), the master pacemaker controlling circadian rhythmicity. Membrane hyperpolarization, caused by lowering the extracellular concentration of potassium or blocking Ca²⁺ influx in SCN cultures by lowering [Ca²⁺], reversibly abolished the rhythmic expression of Per1. In addition, the amplitude of Per1 expression was markedly decreased by voltage-gated Ca²⁺ channel antagonists. A similar result was observed for mouse Per1 and PER2. Together, these results strongly suggest that a transmembrane Ca²⁺ flux is necessary for sustained molecular rhythmicity in the SCN. We propose that periodic Ca²⁺ influx, resulting from circadian variations in membrane potential, is a critical process for circadian pacemaker function.