The pineal gland regulates circadian rhythm through the synthesis and secretion of melatonin. The rise of intracellular Ca²⁺ concentration ([Ca²⁺]_i) following nicotinic acetylcholine receptor (nAChR) stimulation due to parasympathetic nerve activity downregulates melatonin production. Important characteristics and roles of Ca²⁺ mobilization due to nAChR stimulation remain to be clarified. We report here that spontaneous Ca²⁺ oscillations can be observed in approximately 15% of the pinealocytes in slice preparations from rat pineal glands when this dissociation procedure is done within 6 h from a dark-to-light change. The frequency and half-life of [Ca²⁺]_i rise were 0.86 min-1 and 19 s, respectively. Similar spontaneous Ca²⁺ oscillations were recorded in 17% of rat pinealocytes that was primary-cultured for several days. Simultaneous measurement of [Ca²⁺]_i and membrane potential revealed that spontaneous Ca²⁺ oscillations were triggered by periodic membrane depolarizations. Spontaneous Ca²⁺ oscillations in cultured pinealocytes were abolished by extracellular Ca²⁺ removal or application of nifedipine, a blocker of voltage-dependent Ca²⁺ channel (VDCC). In contrast, blockers of intracellular Ca²⁺-release channels, 2-aminoethoxydiphenylborate and ryanodine have no effect. Our results also reveal that, in 23% quiescent pinealocytes, Ca²⁺ oscillations were observed following the withdrawal of nicotine. Norepinephrine-induced melatonin secretion from whole pineal glands was significantly decreased by the co-application of acetylcholine (ACh). This inhibitory effect of ACh was attenuated by nifedipine. In conclusion, both spontaneous and evoked Ca²⁺ oscillations, are due to membrane depolarization following activation of VDCCs. This consists of VDCC 1F subunit, and the associated Ca²⁺ influx can strongly regulate melatonin secretion in pineal glands.