Serotonin (5-HT) caused immediate elevation of intracellular Ca2+ concentration ([Ca2+]i) in blood platelets, and it was completely inhibited by 1 mM EGTA. In Ca2+ replenished platelets, however, 2 mM EGTA did not affect the 5-HT induced elevation of [Ca2+]i when EGTA was applied just before or during the stimulation by 5-HT. At the same concentration 5-HT was also found to enhance Ca2+ influx through the activation of 5-HT2 receptor, but with rather longer latent time. From these results it is suggested that 5-HT induced elevation of [Ca2+]i is caused by mobilization of Ca2+ from intracellular Ca2+ storage sites, but not by direct stimulation of Ca2+ influx. Depletion of such Ca2+ stores might impair the effect of 5-HT on [Ca2+]i. Thus, 5-HT induced augmentation of Ca2+ influx might be secondary to replenishment of the depleted Ca2+ stores which was caused by 5-HT induced internal release of Ca2+. It is concluded that the effects of 5-HT on [Ca2+]i and Ca2+ influx in platelets are manifested sequentially or independently.