Histamine-releasing factors (HRFs) are soluble mediators that can release histamine and other mediators from basophils and mast cells and their activity can vary, depending on the type of IgE. The activity of HRFs is affected by the presence of IgE, although HRF is thought to bind to a specific receptor other than IgE. Until now, HRF signaling pathway including its receptor remains unclear in spite of numerous studies. Since there had been many reports about reactive oxygen species (ROS) as a signaling molecule rather than as a by-product of metabolism, we investigated the possibility of ROS as an intracellular messenger involved in HRF-mediated histamine degranulation. In RBL-2H3 cells, ROS was generated by HRF using H2O2- sensitive fluorescence of fluorescent 2′, 7′-dichlorofluorescein (H2DCFDA). These effects were blocked by anti-oxidant N-acetylcysteine (NAC). These results suggest that ROS generation could play a role as an intracellular messenger in histamine release by HRF.