Intravital microscopy (IVM) is considered as the gold standard for in vivo investigations of dynamic microvascular regulation. The availability of transgenic and knockout animals has propelled the development of murine IVM models for various organs, but technical approaches to the pulmonary microcirculation are still scarce. In anesthetized and ventilated Balb/c mice, we established a microscopic access to the surface of the right upper lung lobe by surgical excision of a window of 7-10 mm diameter from the right thoracic wall. The window was covered by a transparent polyvinylidene membrane and sealed with -cyanoacrylate. Removal of intrathoracic air via a transdiaphragmal intrapleural catheter coupled the lung surface to the window membrane. IVM preparations were hemodynamically stable for at least 120 min with mean arterial blood pressure above 70 mmHg and mean P_aO₂ and P_aCO₂ in the range of 90-100 mmHg and 30-40 mmHg, respectively. Imaged lungs did not show any signs of acute lung injury or edema. Following infusion of FITC dextran, subpleural pulmonary arterioles and venules of up to 50 µm diameter and alveolar capillary networks could be visualized during successive expiratory plateau phases over a period of at least 2 h. Vasoconstrictive responses to hypoxia (11% O₂) or infusion of the thromboxane analog U46619 were prominent in medium-sized arterioles (30 - 50 µm diameter), minor in small arterioles < 30 µm, and absent in venules. The presented IVM model may constitute a powerful new tool for investigations of pulmonary microvascular responses in mice.