Intravital microscopy of the pulmonary microcirculation in research animals is of great scientific interest for its utility in identifying regional changes in pulmonary microcirculatory blood flow. Although feasibility studies have been reported, the pulmonary window can be further refined into a practical tool for pharmaceutical research and drug development. We have established a method to visualize and quantify dynamic changes in three key features of lung function: microvascular red blood cell velocity, flow direction, and hemoglobin saturation. These physiological parameters were measured in an acute closed-chest pulmonary window, which allows real-time images to be captured by fluorescence and multispectral absorption microscopy; images were subsequently quantified using computerized analysis. We validated the model by quantifying changes in microcirculatory blood flow and hemoglobin saturation in two ways: 1) after changes in inspired oxygen content and 2) after pharmacological reduction of pulmonary blood flow via treatment with the β1 adrenergic receptor blocker metoprolol. This robust and relatively simple system facilitates pulmonary intravital microscopy in laboratory rats for pharmacological and physiological research.