Together with cAMP, calcium ions play an important role in the regulation of melatonin synthesis in the pineal organ of all vertebrate species, irrespective of the conspicuous phylogenetic transformation of the melatonin-producing cell, the pinealocyte. Here we address the question how the intracellular concentration of free calcium ions [Ca2+]i is regulated in directly light-sensitive trout pinealocytes and in rat pinealocytes which have lost the direct light sensitivity and respond to norepinephrine. Isolated pinealocytes identified by the S-antigen immunoreaction were investigated by means of the fura-2 technique, image analysis and patch clamp recordings. Approximately 30% of the trout pinealocytes exhibited spontaneous [Ca2+]i oscillations that were not affected by light or dark adaptation of the cells. Removal of extracellular Ca2+ or application of 10 microM nifedipine caused a reversible breakdown of the [Ca2+]i oscillations. Treatments with 60 mM KCl and nifedipine suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca2+]i in both oscillating and nonoscillating trout pinealocytes. Experiments with thapsigargin (2 microM) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role in the regulation of [Ca2+]i remains elusive. Norepinephrine had no apparent effect on [Ca2+]i in any trout pinealocyte. In rat pinealocytes, [Ca2+]i did not show spontaneous oscillations. Norepinephrine evoked a dramatic biphasic rise in [Ca2+]i in more than 95% of the cells via stimulation of alpha1-adrenergic receptors. The response reflects a combination of calcium mobilization from intracellular, thapsigargin-sensitive calcium stores and an increased calcium influx. Voltage-gated calcium channels of the L-type are present in the rat pinealocyte membrane, but they are not involved in the norepinephrine-induced calcium response. These channels, however, mediate the increase in calcium influx which is observed in virtually all rat pinealocytes upon stimulation with acetylcholine or nicotine. The results show that the mechanisms which regulate [Ca2+]i in pinealocytes are complex and differ considerably between poikilothermic and mammalian species.