In vitro differentiation of mouse embryonic stem cells within three-dimensional cell aggregates called embryoid bodies parallels the development of postimplantation embryos at the egg cylinder stage, where visceral and parietal endoderm diverge from the primitive endoderm. We have investigated spontaneous [Ca2+]i oscillations by means of confocal laser-scanning microscopy in primitive endodermal cell layers of embryoid bodies during their differentiation to parietal and visceral endoderm. The frequency of [Ca2+]i oscillations increased from day 4 to day 19 of development, whereas their duration decreased from day 3 to days 16-17. Oscillations depended on both extracellular Ca2+ and Ca2+ release from intracellular stores as they were abolished in Ca(2+)-free solution and in the prescence of Ni2+ and thapsigargin. Signal transduction operated via the phospholipase C (PLC)-mediated inositol 1,4,5-triphosphate (InsP3) pathway with a negative feedback loop via protein kinase C (PKC) as U73,122, a blocker of PLC; bisindolylmaleimide 1, staurosporine, and H-7, blockers of PKC; and 10 mM caffeine totally inhibited [Ca2+]i spiking. Thimerosal, which hypersensitizes the InsP3 receptor, as well as vasopressin and bradykinin, which act via the InsP3 pathway, increased the frequency of [Ca2+]i spikes. In the prescence of brefeldin A (50 microM) or monensin (20 microM), which both inhibit endo/exocytotic vesicle pathways, an immediate transient increase in spiking activity was followed by a decline within 1 to 2 h. In the presence of brefeldin A or thapsigargin or in the absence of extracellular Ca2+, endocytotic vesicles were absent, suggesting that oscillating [Ca2+]i transients are involved in the exo/endocytotic vesicle shuttle.