Lysophosphatidic acid (LPA) is one of the major mitogens found in blood serum. This bioactive phospholipid mediates various cellular responses through G-protein coupled transmembrane receptors. In many cell types LPA is known to trigger an increase in intracellular Ca²⁺ concentration, which is involved in the regulation of cell proliferation. The control of the proliferative state in embryonic stem (ES) cells is of high significance in view of their potential clinical use. However, the signalling pathways involved in the maintenance of this state are still not fully understood. In the present study we investigated whether LPA, as an extracellular molecule, provoked an effect on Ca²⁺ mobilization in D3 mouse ES cells and studied the molecular mechanisms involved. Using the Fura-2 fluorescence technique we demonstrated that indeed LPA over the range of 100 nM to 10 µM activates Ca²⁺ signalling in 96% of the mouse ES cells examined (n=28 experiments). This Ca²⁺ signal was mediated by mobilization from internal stores given that Ca²⁺ release was reproduced in the absence of extracellular Ca²⁺ (n=10 experiments). Consisting with this finding, treating the cells with 1µM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca²⁺ ATPase, completely abolished the LPA-stimulated Ca²⁺ increase in 3 out of 4 assays. Furthermore, we confirmed that in this LPA-induced Ca²⁺ mobilization the inositol 1,4,5-triphosphate (InsP3) activation was involved as the specific phospholipase C (PLC) inhibitor U73122 (3 µM) completely attenuated the increase in calcium concentration (n=6). On the contrary, the inactive analogue U-73343 applied at the same concentration (3 µM) did not produce any effect (n=4). In addition, we saw that LPA effect could be exerted through at least two types of receptors, LPA-1 and LPA-2 whose mRNAs were detected by RT-PCR assays (n=4). On a next stage we shall focus on the effect of this calcium mobilization on ES cell proliferation, investigating the molecular mechanisms involved. The basic research in the area of stem cells will contribute to a better understanding of their proper characteristics and signal pathways, as well as proliferation and differentiation processes assuring their better clinical application.