OBJECTIVE The aim of this study was to determine whether diacylglycerol kinase (DGK) is involved in transplasmalemmal Ca²⁺ influx of platelets. METHODS Effects of R59949, an inhibitor of diacylglycerol kinase, on intracellular Ca²⁺ concentration ([Ca²⁺](i) ) and mRNA expression of DGK isozymes were investigated using washed human platelet suspensions. RESULTS Thrombin-induced increase in [Ca²⁺](i) was significantly inhibited by pretreatment of platelets with R59949, while thapsigargin-induced increase in [Ca²⁺](i) was comparable in platelets with and without R59949 pretreatment. Thapsigargin-induced increase in [Ca²⁺](i) was markedly attenuated in the presence of SKF-96365. In the presence of SKF-96365, thrombin-induced increase in [Ca²⁺](i) was significantly attenuated, and additional treatment with R59949 caused a further decrease in [Ca²⁺](i) . Pretreatment of platelets with 1-butanol significantly attenuated thrombin-induced increase in [Ca²⁺](i) , while thrombin-induced increase in [Ca²⁺](i) was augmented in the presence of propranolol. mRNA expression of DGK-α and DGK-γ, which are known to be inhibited by R59949, in platelets was confirmed by RT-PCR analysis. CONCLUSIONS R59949 inhibited a store-depletion-insensitive component of transplasmalemmal Ca²⁺ entry induced by thrombin, while store-operated Ca²⁺ entry was not affected by R59949. The results of this study suggest that phosphatidic acid is involved in thrombin-induced Ca²⁺ influx of platelets.