Chlorine (Cl₂) inhalation induces severe oxidative lung injury and airway hyperresponsiveness (AHR) that lead to asthmalike symptoms. When inhaled, Cl₂ reacts with epithelial lining fluid, forming by-products that damage hyaluronan, a constituent of the extracellular matrix, causing the release of low-molecular-weight fragments (L-HA, <300 kDa), which initiate a series of proinflammatory events. Cl₂ (400 ppm, 30 min) exposure to mice caused an increase of L-HA and its binding partner, inter-α-trypsin-inhibitor (IαI), in the bronchoalveolar lavage fluid. Airway resistance following methacholine challenge was increased 24 h post-Cl₂ exposure. Intratracheal administration of high-molecular-weight hyaluronan (H-HA) or an antibody against IαI post-Cl₂ exposure decreased AHR. Exposure of human airway smooth muscle (HASM) cells to Cl₂ (100 ppm, 10 min) or incubation with Cl₂-exposed H-HA (which fragments it to L-HA) increased membrane potential depolarization, intracellular Ca²⁺, and RhoA activation. Inhibition of RhoA, chelation of intracellular Ca²⁺, blockade of cation channels, as well as postexposure addition of H-HA, reversed membrane depolarization in HASM cells. We propose a paradigm in which oxidative lung injury generates reactive species and L-HA that activates RhoA and Ca²⁺ channels of airway smooth muscle cells, increasing their contractility and thus causing AHR.