Emerging evidence suggests that Ca2+ signals are important for the self-renewal and differentiation of human embryonic stem cells (hESCs).However,little is known about the physiological and pharmacological properties of the Ca2+-handling machinery in hESCs.In this study we used RT-PCR and Weste blotting to analyze the expression profiles of genes encoding Ca2+-handling proteins;we also used confocal Ca2+ imaging and pharmacological approaches to determine the contribution of the Ca2+-handling machinery to the regulation of Ca2+ signaling in hESCs.We revealed that hESCs expressed pluripotent markers and various Ca2+-handling-related genes.ATP-induced Ca2+ transients in almost all hESCs were inhibited by the inositol-1,4,5-triphosphate receptor (IP3R) blocker 2-APB or xestospongin C.In addition,Ca2+ transients were induced by a ryanodine receptor (RyR) activator,caffeine,in 10％-15％ of hESCs and were blocked by ryanodine,whereas caffeine and ATP did not have additive effects.Moreover,store-operated Ca2+ entry (SOCE) but not voltage-operated Ca2+ channel-mediated Ca2+ entry was observed.Inhibition of sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA) by thapsigargin induced a significant increase in the cytosolic free Ca2+ concentration ([Ca2+]i).For the Ca2+ extrusion pathway,inhibition of plasma membrane Ca2+ pumps (PMCAs) by carboxyeosin induced a slow increase in [Ca2+]i,whereas the Na+/Ca2+ exchanger (NCX) inhibitor KBR7943 induced a rapid increase in [Ca2+]i.Taken together,increased [Ca2+]i iS mainly mediated by Ca2+ release from intracellular stores via IP3Rs.In addition,RyRs function in a portion of hESCs,thus indicating heterogeneity of the Ca2+-signaling machinery in hESCs;maintenance of low [Ca2+]i is mediated by uptake of cytosolic Ca2+ into the ER via SERCA and extrusion of Ca2+ out of cells via NCX and PMCA in hESCs.