In this pilot study, algorithms for quantitatively evaluating the distribution and heterogeneity of human ventilation imaged with hyperpolarized (HP) ³He MRI were developed for the goal of examining structure-function relationships within the asthmatic lung. Ten asthmatic and six healthy human subjects were imaged with HP ³He MRI before bronchial challenge (preMCh), after bronchial challenge (postMCh), and after a series of deep inspirations (DI) (postDI) following challenge. The acquired images were rigidly coregistered. Local voxel fractional ventilation was computed from setting the sum of the pixel intensity within the lung region in each image set to 1 liter of inhaled ³He mixture. Local ventilation heterogeneity was quantified by computing regional signal coefficient-of-variation. Voxel fractional ventilation histograms and overall heterogeneity scores were then calculated. Asthmatic subjects had a higher ventilation heterogeneity to begin with (p=0.025). A methacholine challenge elevated ventilation heterogeneity for all subjects (difference p=0.08). After a DI post-challenge, this heterogeneity reversed substantially towards the baseline state for healthy subjects, but only minimally in asthmatic subjects. This difference was significant both in absolute quantity (difference p=0.007) as well as relative to the initial increase (difference p=0.03). These findings suggest that heterogeneity is not a characteristic unique to asthma, but rather a behavior intrinsic to all airway trees when provoked. Once ventilation heterogeneity is established, it is the lack of reversal following DIs that distinguishes asthmatics from non-asthmatics.