The regional pattern and extent of airway closure measured by three-dimensional ventilation imaging may relate to airway hyperresponsiveness (AHR) and peripheral airways disease in asthmatic subjects. We hypothesized that asthmatic airways are predisposed to closure during bronchoconstriction in the presence of ventilation heterogeneity and AHR. Fourteen asthmatic subjects (6 women) underwent combined ventilation single photon emission computed tomography/computed tomography scans before and after methacholine challenge. Regional airway closure was determined by complete loss of ventilation following methacholine challenge. Peripheral airway disease was measured by multiple-breath nitrogen washout from which S(cond) (index of peripheral conductive airway abnormality) was derived. Relationships between airway closure and lung function were examined by multiple-linear regression. Forced expiratory volume in 1 s was 87.5 ± 15.8% predicted, and seven subjects had AHR. Methacholine challenge decreased forced expiratory volume in 1 s by 23 ± 5% and increased nonventilated volume from 16 ± 4 to 29 ± 13% of computed tomography lung volume. The increase in airway closure measured by nonventilated volume correlated independently with both S(cond) (partial R(2) = 0.22) and with AHR (partial R(2) = 0.38). The extent of airway closure induced by methacholine inhalation in asthmatic subjects is greater with increasing peripheral airways disease, as measured by ventilation heterogeneity, and with worse AHR.