Reactive oxygen species (ROS) are known to modulate activities of a host of kinases, phosphatases and transcription factors. Rutin and chlorogenic acid (CGA) are the major polyphenolic antioxidants present in the small molecular fraction of smokeless tobacco leaf extracts, as ascertained by reverse-phase high-pressure liquid chromatography (HPLC) and mass spectrometry. Levels of intracellular ROS in resting versus antigen-immunoglobulin E (IgE)-challenged murine mast cells were measured at 510 nm by fluorescence-activated cell sorting (FACS) using carboxy-dichlorofluorescein (DCFH-DA). Enhanced ROS production was observed in IgE-sensitized mast cells following antigenic challenge. Rutin and CGA reduced ROS levels in antigen-IgE-activated mast cells. Concomitantly, they also profoundly inhibited histamine release by these activated mast cells. In contrast, rutin and CGA augmented the inducible cytokine messages, i.e. interleukin (IL)-10, IL-13, interferon-gamma (IFN-gamma), IL-6 and tumour necrosis factor-alpha (TNF-alpha) in IgE-sensitized mast cells following antigen challenge. This study indicates that tobacco polyphenolic antioxidants that quench intracellular ROS, differentially affect two effector functions of antigen-IgE-activated mast cells. This model system may be employed to determine the molecular target of polyphenols. The potential role of these polyphenolic antioxidants on IgE-mediated allergy in vivo depends on a balance of their differential effects on mast cell activation.