Reactive oxygen species (ROS) are produced in various cells and affect many biologic processes. In this study, we examined the effects of 2-methyl-1,4-naphtoquinone (menadione; vitamin K3) on signal transduction in mast cells. Several lines of evidence suggest that H2O2 affects the antigen-induced responses in mast cells but its mechanism is not clearly understood. Unlike H2O2, menadione produces ROS only inside cells. Thus, it is possible to investigate the effects of ROS produced intracellularly. Pretreatment of mast cells (RBL-2H3) with menadione inhibited exocytotic secretion (degranulation) induced by antigen stimulation dose dependently. Menadione also inhibited the intracellular Ca2+ increase induced by antigen stimulation. Menadione did not inhibit the Ca2+ increase due to Ca2+ release from the intracellular calcium store in the absence of extracellular Ca2+, but inhibited the Ca2+ influx from the extracellular medium. These results suggest that reactive oxygen generated inside RBL cells by menadione inhibited degranulation by decreasing Ca2+ influx through the store operated Ca2+ channel on the plasma membrane.