Cerebellar Purkinje neurons contain a remarkable array of cellular components potentially concerned with regulation of the free cytoplasmic Ca2+ concentration, [Ca2+]i. These include high concentrations of Ca(2+)-binding proteins, inositol 1,4,5-triphosphate receptors (IP3R), and ryanodine receptors (RyR). The latter two molecules are thought to be associated with intracellular Ca2+ stores. We have examined the properties of such stores in cultured rat cerebellar neurons taken from 16 d rat embryos. In this system, about half of the neurons could be identified as Purkinje-like cells, as indicated by staining for the Ca(2+)-binding protein calbindin D-28k, as well as for IP3R and RyR. In double immunofluorescent staining, the IP3R and RyR immunoreactivity primarily colocalized with the staining for calbindin. The cells responded to glutamate, kainate, and quisqualate with large increases in the somatic [Ca2+]i but failed to respond directly to NMDA (10-50 microM). Furthermore, the neurons expressed active membrane conductances, repetitive action potential firing, and spontaneous firing patterns similar to those reported for cerebellar Purkinje neurons in vivo. Action potential firing produced changes in somatic [Ca2+]i that were quite small or absent in most cells. However, blocking spike repolarization with tetraethylammonium (5 mM) produced substantial transient elevations in somatic [Ca2+]i, suggesting the expression of some Ca2+ channels in the somatic membrane. Caffeine (10 mM) released Ca2+ from intracellular stores in about one-half of the cultured neurons. This effect could be repeated if the stores were first reloaded by a depolarization-induced elevation in [Ca2+]i. The effects of caffeine were reduced by prolonged application of ryanodine (10 microM). We were also able to demonstrate that the caffeine-sensitive Ca2+ stores could regulate electrophysiological events in some cells, altering patterns of spontaneous activity. Furthermore, in the presence of caffeine, [Ca2+]i signals induced by an evoked spike train were larger and accompanied by long-lasting after hyperpolarizations. We conclude that in addition to providing a releasable pool of Ca2+, the caffeine-sensitive stores also influence cellular events by their contribution to Ca2+ buffering.