PURPOSE: To develop and demonstrate a method for regional evaluation of pulmonary perfusion and gas exchange based on intravenous injection of hyperpolarized xenon 129 ((129)Xe) and subsequent magnetic resonance (MR) imaging of the gas-phase (129)Xe emerging in the alveolar airspaces. MATERIALS AND METHODS: Five Fischer 344 rats that weighed 200-425 g were prepared for imaging according to an institutional animal care and use committee-approved protocol. Rats were ventilated, and a 3-F catheter was placed in the jugular (n = 1) or a 24-gauge catheter in the tail (n = 4) vein. Imaging and spectroscopy of gas-phase (129)Xe were performed after injecting 5 mL of half-normal saline saturated with (129)Xe hyperpolarized to 12%. Corresponding ventilation images were obtained during conventional inhalation delivery of hyperpolarized (129)Xe. RESULTS: Injections of (129)Xe-saturated saline were well tolerated and produced a strong gas-phase (129)Xe signal in the airspaces that resulted from (129)Xe transport through the pulmonary circulation and diffusion across the blood-gas barrier. After a single injection, the emerging (129)Xe gas could be detected separately from (129)Xe remaining in the blood and was imaged with an in-plane resolution of 1 x 1 mm and a signal-to-noise ratio of 25. Images in one rat revealed a matched ventilation-perfusion deficit, while images in another rat showed that xenon gas exchange was temporarily impaired after saline overload, with recovery of function 1 hour later. CONCLUSION: MR imaging of gas-phase (129)Xe emerging in the pulmonary airspaces after intravenous injection has the potential to become a sensitive and minimally invasive new tool for regional evaluation of pulmonary perfusion and gas exchange. SUPPLEMENTAL MATERIAL: http://radiology.rsnajnls.org/cgi/content/full/2513081550/DC1.