Background: Contraction of bronchial smooth muscle is the main mechanism of asthmatic responses. Inflammatory cells such as eosinophils and mast cells produce chemical mediators that induce smooth muscle contraction. To investigate the mechanisms of IgE-independent asthmatic response in murine asthma models, a novel in vitro assay system using primary cultured mouse bronchial smooth muscle cells (BSMC) was explored. Method: Trachea and bronchi were taken from young mice and underwent primary culture. BSMC were expanded in culture and then embedded in a collagen gel. The well-known constrictors leukotriene D₄ (LTD₄), methacholine and histamine were applied to the BSMC gels. The gel images were captured by an image analyzer and contractile responses were evaluated. Results: LTD₄ and methacholine significantly induced the gel contraction in a dose-dependent manner, but histamine did not. Montelukast, a CysLT type ΙΙ receptor antagonist, and atropine, a muscarinic acetylcholine receptor antagonist, inhibited the contractile responses in an agonist-specific manner. Conclusion: A contraction assay system using cultured mouse BSMC was successfully established for the first time. It may go a long way toward identifying bronchoconstricting mediators involved in murine asthma models.