The purpose of this work is to investigate the use of dual-energy microcomputed tomography (CT) for the estimation of vascular, tissue, and air fractions in rodent lungs using a postreconstruction three material decomposition method. Using simulations, we have estimated the accuracy limits of the decomposition for realistic micro-CT noise levels. Next, we performed experiments involving ex vivo lung imaging in which intact rat lungs were carefully removed from the thorax, injected with an iodine-based contrast agent, and then inflated with different volumes of air (n ϭ 2). Finally, we performed in vivo imaging studies in C57BL/6 mice (n ϭ 5) using fast prospective respiratory gating in end inspiration and end expiration for three different levels of positive end expiratory pressure (PEEP). Before imaging, mice were injected with a liposomal blood pool contrast agent. The three-dimensional air, tissue, and blood fraction maps were computed and analyzed. The results indicate that separation and volume estimation of the three material components of the lungs are possible. The mean accuracy values for air, blood, and tissue were 93, 93, and 90%, respectively. The absolute accuracy in determining all fraction materials was 91.6%. The coefficient of variation was small (2.5%) indicating good repeatability. The minimum difference that we could detect in material fractions was 15%. As expected, an increase in PEEP levels for the living mouse resulted in statistically significant increases in air fractions at end expiration but no significant changes at end inspiration. Our method has applicability in preclinical pulmonary studies where changes in lung structure and gas volume as a result of lung injury, environmental exposures, or drug bioactivity would have important physiological implications. micro-computed tomography; small animal imaging; perfusion COMPUTED TOMOGRAPHY (CT) IS one of the most used imaging modalities for the evaluation of thoracic disorders. With the recently developed dual-energy CT technique (DECT), the clinical utility of CT in the pulmonary diseases could expand even more. DECT allows analysis of the chemical composition of tissues by means of dual-energy data acquisition and tissue decomposition (14). CT data analysis with DECT is based on differences in X-ray absorption of heavy elements with energy. In clinical use, many DECT studies are already focusing on the thorax (9,18,19,(23)(24)(25)(26)(27). In the thorax, the three materials most frequently analyzed are iodine, air, and soft tissue. Recently, researchers (26) have evaluated the feasibility of using DECT for pulmonary perfusion and ventilation in the lungs using both iodinated contrast agents and xenon to provide distributions representing the local perfusion and ventilation.More interestingly, a recent study (8) has abandoned xenon for ventilation imaging in CT because the use of xenon gas is logistically demanding and requires the use of special inhalators and pressurized gas bottles. Consequently, the authors have investigated the fe...