The alveolated structure of the pulmonary acinus plays a vital role in gas exchange function. Three-dimensional (3D) analysis of the parenchymal region is fundamental to understanding this structure-function relationship, but only a limited number of attempts have been conducted in the past because of technical limitations. In this study, we developed a new image processing methodology based on finite element (FE) analysis for accurate 3D structural reconstruction of the gas exchange regions of the lung. Stereologically well characterized rat lung samples (Pediatr Res 53: 72–80, 2003) were imaged using high-resolution synchrotron radiation-based X-ray tomographic microscopy. A stack of 1,024 images (each slice: 1024 × 1024 pixels) with resolution of 1.4 μm³ per voxel were generated. For the development of FE algorithm, regions of interest (ROI), containing ∼7.5 million voxels, were further extracted as a working subunit. 3D FEs were created overlaying the voxel map using a grid-based hexahedral algorithm. A proper threshold value for appropriate segmentation was iteratively determined to match the calculated volume density of tissue to the stereologically determined value (Pediatr Res 53: 72–80, 2003). The resulting 3D FEs are ready to be used for 3D structural analysis as well as for subsequent FE computational analyses like fluid dynamics and skeletonization.