Our goal was to develop a method for generating high-resolution three-dimensional pulmonary compliance images in rodents from computed tomography (CT) images acquired at a series of constant pressures in ventilated animals. One rat and one mouse were used to demonstrate this technique. A pre-clinical GE flat panel CT scanner (maximum 31 line-pairs cm(-1) resolution) was utilized for image acquisition. The thorax of each animal was imaged with breath-holds at 2, 6, 10, 14 and 18 cm H2O pressure in triplicate. A deformable image registration algorithm was applied to each pair of CT images to map corresponding tissue elements. Pulmonary compliance was calculated on a voxel by voxel basis using adjacent pairs of CT images. Triplicate imaging was used to estimate the measurement error of this technique. The 3D pulmonary compliance images revealed regional heterogeneity of compliance. The maximum total lung compliance measured 0.080 (+/-0.007) ml air per cm H2O per ml of lung and 0.039 (+/-0.004) ml air per cm H2O per ml of lung for the rat and mouse, respectively. In this study, we have demonstrated a unique method of quantifying regional lung compliance from 4 to 16 cm H2O pressure with sub-millimetre spatial resolution in rodents.