Acinar geometry has been the subject of several morphological and imaging studies in the past; however, surprisingly little is known about how the acinar microstructure changes when the lung inflates or deflates. Lung morphometry with hyperpolarized (3)He diffusion MRI allows non-destructive evaluation of lung microstructure and acinar geometry, which has important applications in understanding basic lung physiology and disease. In this study, we have measured the alveolar and acinar duct sizes at physiologically relevant volumes by (3)He lung morphometry in six normal, excised, and unfixed canine lungs. Our results imply that, during a 37% decrease in lung volume, the acinar duct radius decreases by 19%, whereas the alveolar depth increases by 9% (P < 0.0001 and P < 0.05, respectively via paired t-tests with a Bonferroni correction). A comparison to serial sections under the microscope validates the imaging results and opens the door to in vivo human studies of lung acinar geometry and physiology during respiration using (3)He lung morphometry.