Intravital microscopy (IVM) is the gold standard for in vivo investigations of the microcirculation. The availability of transgenic animals has propelled the development of murine IVM models for various organs, but not yet for the lung. Here, we report a new IVM model for studies of the murine pulmonary microcirculation. In mechanically ventilated Balb/c mice, a window was surgically excised from the right thoracic wall providing visual access to the upper lobe of the right lung. The window was sealed with a transparent polyvinylidene membrane and cyanoacrylate. Intrathoracic air was removed by a transabdominal intrapleural catheter. IVM preparations were hemodynamically stable with mean arterial pressures >70mmHg and physiological arterial blood gases for >2hrs. Wet/dry ratio of imaged lungs did not differ from control lungs. Within each window of observation, 4-5 arterioles and 2-3 venules with diameters ranging between 56–20mm and 34–16mm could be stably visualized. By use of FITC dextran as a plasma marker, we evaluated hypoxic pulmonary vasoconstriction (PVC) in this model. Within 10 min, hypoxic ventilation with 11% O₂ reduced vascular diameters in medium-sized arterioles (30–50mm) by 6.6±0.84%, but did not cause PVC in small-sized arterioles (<30mm) or venules. No further reduction of vessel diameters was observed within 1 hr. This model presents a powerful new technique for studies of the pulmonary microcirculation.