Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca(2+) concentration ([Ca(2+)]i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca(2+), in this calcium hypothesis, serves as a reservoir of Ca(2+) source. Recently we find that extracellular Ca(2+)per se inhibits the [Ca(2+)]i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca(2+) or both [1]. In this work we showed that, in physiological condition, extracellular Ca(2+)per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca(2+) in physiological concentration (2.5mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca(2+) directly triggered vesicle exocytosis without eliciting intracellular Ca(2+). We propose that intracellular Ca(2+) and extracellular Ca(2+)per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca(2+), while the vesicle quantal size was mainly determined by extracellular Ca(2+) in chromaffin cells physiologically.