Prostaglandin D₂ (PGD₂) is one of the key lipid mediators of allergic airway inflammation, including bronchial asthma. However, the role of PGD₂ in the pathogenesis of asthma is not fully understood. In the present study, the effect of PGD₂ on smooth muscle contractility of the airways was determined to elucidate its role in the development of airway hyperresponsiveness (AHR). In isolated bronchial smooth muscles (BSMs) of naive mice, application of PGD₂ (10⁻⁹–10⁻⁵ M) had no effect on the baseline tension. However, when the tissues were precontracted partially with 30 mM K⁺ (in the presence of 10⁻⁶ M atropine), PGD₂ markedly augmented the contraction induced by the high K⁺ depolarization. The PGD₂-induced augmentation of contraction was significantly inhibited both by 10⁻⁶ M laropiprant (a selective DP₁ antagonist) and 10⁻⁷ M Y-27632 (a Rho-kinase inhibitor), indicating that a DP₁ receptor-mediated activation of Rho-kinase is involved in the PGD₂-induced BSM hyperresponsiveness. Indeed, the GTP-RhoA pull-down assay revealed an increase in active form of RhoA in the PGD₂-treated mouse BSMs. On the other hand, in the high K⁺-depolarized cultured human BSM cells, PGD₂ caused no further increase in cytosolic Ca²⁺ concentration. These findings suggest that PGD₂ causes RhoA/Rho-kinase-mediated Ca²⁺ sensitization of BSM contraction to augment its contractility. Increased PGD₂ level in the airways might be a cause of the AHR in asthma.