Background and Purpose Mast cell hyperplasia has been observed in the lungs of mice with experimental asthma, but few reports have studied basophils. Here, we attempted to discriminate and quantify mast cells and basophils in the lungs in a murine asthma model, determine if both cells were increased by multiple antigen challenges and assess the roles of those cells in asthmatic responses. Experimental Approach Sensitized Balb/c mice were intratracheally challenged with ovalbumin four times. Mast cells and basophils in enzymatically digested lung tissue were detected by flow cytometry. An anti-FcεRI monoclonal antibody, MAR-1, was i.p. administered during the multiple challenges. Key Results The numbers of both mast cells (IgE+ C-kit+) and basophils (IgE+ C-kit- CD49b+) increased in the lungs after three challenges. Treatment with MAR-1 completely abolished the increases; however, a late-phase increase in specific airway resistance (sRaw), and airway eosinophilia and neutrophilia were not affected by the treatment, although the early-phase increase in sRaw was suppressed. MAR-1 reduced antigen-induced airway IL-4 production. Basophils infiltrating the lung clearly produced IL-4 after antigen stimulation in vitro; however, histamine and murine mast cell protease 1 were not increased in the serum after the challenge, indicating that mast cell activation was not evoked. Conclusion and Implications Both mast cells and basophils infiltrated the lungs by multiple intratracheal antigen challenges in sensitized mice. Neither mast cells nor basophils were involved in late-phase airway obstruction, although early-phase obstruction was mediated by basophils. Targeting basophils in asthma therapy may be useful for an early asthmatic response. © 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.