Quantitative in-vivo imaging of lung perfusion in rodents can provide critical information for preclinical studies. However, the combined challenges of high temporal and spatial resolution have made routine quantitative perfusion imaging difficult in rodents. We have recently developed a dual tube/detector micro-CT scanner that is well suited to capture first-pass kinetics of a bolus of contrast agent used to compute perfusion information. Our approach is based on the paradigm that the same time density curves can be reproduced in a number of consecutive, small (i.e. 50μL ) injections of iodinated contrast agent at a series of different angles. This reproducibility is ensured by the high-level integration of the imaging components of our system, with a micro-injector, a mechanical ventilator, and monitoring applications. Sampling is controlled through a biological pulse sequence implemented in LabVIEW. Image reconstruction is based on a simultaneous algebraic reconstruction technique implemented on a GPU. The capabilities of 4D micro-CT imaging are demonstrated in studies on lung perfusion in rats. We report 4D micro-CT imaging in the rat lung with a heartbeat temporal resolution of 140 ms and reconstructed voxels of 88 μm. The approach can be readily extended to a wide range of important preclinical models, such as tumor perfusion and angiogenesis, and renal function.