Aim:To investigate the developmental regulation of intracellular Ca~(2+)transients,an essential event in excitation-contraction coupling,during cardiomyocytedifferentiation.Methods:Using the embryonic stem(ES)cell in vitro differentia-tion system and pharmacological intervention,we investigated the molecular andfunctional regulation of Ca~(2+)handling proteins on the Ca~(2+)transients at early,intermediate and later differentiation stages of ES cell-derived cardiomyocytes(ESCM).Results:Nifedipine,a selective antagonist of L-type Ca~(2+)channels,totally blocked Ca~(2+)transients even in the condition of field-electric stimulation inESCM at three differentiation stages.The Ca~(2+)transients of ESCM were alsoinhibited by both ryanodine[an inhibitor of ryanodine receptors(RyRs)]and 2-aminoethoxydipheylborate[2-APB,an inhibitor of inositol-1,4,5-trisphosphatereceptors(IP_3Rs)].The inhibitory effect of ryanodine increased with the time ofdifferentiation,while the effect of 2-APB decreased with the differentiation.Thapsigargin,an inhibitor of SR Ca~(2+)-pump ATPase,inhibited Ca~(2+)transientsequally at three differentiation stages that matched the expression profile.Na~+free solution,which inhibits Na~+-Ca~(2+)exchanger(NCX)to extrude Ca~(2+)from cytosol,did not affect the amplitude of Ca~(2+)transients of ESCM until the latter differentia-tion stage,but it significantly enhanced the basal Ca~(2+)concentration.Conclusion:The Ca~(2+)transients in ESCM depend on both the sarcolemmal Ca~(2+)entry viaL-type Ca~(2+)channels and the SR Ca~(2+)release from RyRs and IP_3Rs even at theearly differentiation stage;but NCX seems not to regulate the peak of Ca~(2+)tran-sients until the latter differentiation stage. To investigate the developmental regulation of intracellular Ca ~ (2+) transients, an essential event in excitation-contraction coupling, during cardiomyocystifferentiation. Methods: Using the embryonic stem (ES) cell in vitro differentia tion system and pharmacological intervention, we investigated the molecular and functional regulation of Ca ~ (2+) handling proteins on the Ca ~ (2+) transients at early, intermediate and later differentiation stages of ES cell-derived cardiomyocytes (ESCM). Results: Nifedipine, a selective antagonist of L totally blocked Ca ~ (2+) transients even in the condition of field-electric stimulation in ESCM at three differentiation stages. Ca ~ (2+) transients of ESCM were also inhibited by both ryanodine [ an inhibitor of ryanodine receptors (RyRs)] and 2-aminoethoxydipheylborate [2-APB, an inhibitor of inositol-1,4,5-trisphosphate receptors (IP_3Rs)]. The inhibitory effect of ryanodine increased with the time of differentiation, while the effect of 2-APB decreased wi th the differentiation. Actinarigate, an inhibitor of SR Ca ~ (2 +) - pump ATPase, inhibited Ca ~ (2+) transientsequally at three differentiation stages that matched the expression profile. Na ~ + free solution, which inhibits Na ~ + - Ca ~ (2+) exchanger (NCX) to extrude Ca ~ (2+) from cytosol, did not affect the amplitude of Ca ~ (2+) transients of ESCM until the latter differentia- tion stage, but it significantly enhanced the basal Ca 2+ concentration.Conclusion: The Ca 2+ transients in ESCM depend on both the sarcolemmal Ca 2+ entry via L-type Ca 2+ channels and the SR Ca 2+ ) release from RyRs and IP_3Rs even at the early differentiation stage; but NCX seems not to regulate the peak of Ca ~ (2+) tran-sients until the latter differentiation stage.