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In vivo imaging is an important tool for preclinical studies of lung function and disease. The widespread availability of multimodal animal imaging systems and the rapid rate of diagnostic contrast agent development have empowered researchers to noninvasively study lung function and pulmonary disorders. Investigators can identify, track, and quantify biological processes over time. In this review, we highlight the fundamental principles of bioluminescence, fluorescence, planar X-ray, X-ray computed tomography, magnetic resonance imaging, and nuclear imaging modalities (such as positron emission tomography and single photon emission computed tomography) that have been successfully employed for the study of lung function and pulmonary disorders in a preclinical setting. The major principles, benefits, and applications of each imaging modality and technology are reviewed. Limitations and the future …