Abstract: The level of reactive oxygen species (ROS) and the activity of spontaneous, transient, localized Ca2+ increases (known as Ca2+ sparks) in tracheal smooth muscle cells (TSMCs) in an experimental allergic asthma mouse model has not yet been investigated. We used laser confocal microscopy and fluorescent dyes to measure ROS levels and Ca2+ sparks, and we found that both events were significantly increased in TSMCs obtained from ovalbumin (OVA)-sensitized/-challenged mice compared with control mice. ROS levels began to increase in TSMCs after the first OVA challenge, and this increase was sustained. However, this elevation and Ca2+-spark increase was abolished after the administration of the ROS scavenger N-acetylcysteine amide (NACA) for 5 days. Furthermore, a similar inhibition was also observed following the direct perfusion of NACA into cells isolated from the (OVA)-sensitized mice that were not treated with NACA. Moreover, we used 0.1-mM caffeine treatment to increase the Ca2+ sparks in single TSMCs and observed cell shortening. In addition, we did not find increases in the mRNA levels of ryanodine (RyRs) and inositol 1,4,5-trisphosphate (IP(3)Rs) receptors in the tracheal smooth muscle cells of (OVA)-sensitized mice compared with controls. We concluded that ROS and Ca2+ sparks increased in (OVA)-sensitized TSMCs. We found that ROS induces Ca2+ sparks, and increased Ca2+ sparks resulted in the contraction of (OVA)-sensitized TSMCs, resulting in the generation of airway hyperresponsiveness (AHR). This effect may represent a novel mechanism for AHR pathogenesis and might provide insight into new methods for the clinical prevention and treatment of asthma and asthmatic AHR.