To clarify the role of lymphocytes and eosinophils in the late asthmatic response (LAR) observed several hours after OA challenge, and in airway inflammation observed 24 hrs after OA challenge in OA sensitized guinea pigs, we examined the infiltration and/or accumulation of lymphocytes and eosinophils in lung tissues by PKH2 labeling technique and Luna staining technique, respectively. The guinea pigs showed LAR 6 hrs after exposure to antigen, when moderate infiltration of lymphocytes and marked accumulation of eosinophils were observed in lung tissues. At 24 hrs after OA challenge, intensive infiltrations of both lymphocytes and eosinophils into the lung were observed. Pretreatment with MTX (methotrexate) suppressed LAR, inhibited infiltrations of lymphocytes and eosinophils into lung tissues at 6 hrs (LAR) and at 24 hrs after OA challenge, and prevented bronchial hyperresponsiveness to methacholine 48 hrs after antigen exposure in a dose-dependent manner. These findings suggest that lymphocytes and eosinophils are involved in LAR and also in the chronic airway inflammation which is believed to make the bronchial asthmatic condition more severe and more refractory. Also, our results showed that MTX, which has an inhibitory action on such inflammation, may be a useful bronchial asthma.