The pulmonary airways are subdivided into conducting and gas-exchanging airways. The small tree of gas-exchanging airways which is fed by the most distal conducting airway represents an acinus. Very little is known about the development of the number of acini. The goal of this study was to estimate their number throughout rat postnatal development. Right middle rat lung lobes were obtained at postnatal day 4-60, stained with heavy metals, paraffin embedded, and scanned by synchrotron radiation based X-ray tomographic microscopy or imaged using micro computed tomography after critical point drying. The acini were counted by detection of the transitional bronchioles (bronchioalveolar duct junction; BADJ) using morphological criteria (thickness of the walls of airways and appearance of alveoli) during examination of the resulting 3D image stacks. Between postnatal days 4-60, the number of acini per lung remained constant (5840 ± 547 acini), but their volume increased significantly. We conclude that the acini are formed before the end of the saccular stage (before postnatal day 4) and that the developmental increase of the lung volume is achieved by an increase of the acinar volume and not by an increase of their number. Furthermore, our results propose that the bronchioalveolar stem cells, which are residing in the BADJ, are as constant in their location at the BADJ itself.