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; vitamine 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.