Oxygen transport from the blood to the tissues is a diffusive process driven by the gradient of oxygen tension (PO2). We developed an oxygen-quenching fluorescent membrane that allowed visualization of the PO2 distribution near the microvessels as optical patterns on the membrane by epifluorescence microscopy. This membrane was highly gas permeable to allow PO2 measurement and was transparent enough to also permit observation of the microcirculation. In combination with a newly devised gastight chamber and a micropositioning system, this membrane technique made it possible to visualize the PO2 distribution in the rat mesenteric microvascular network under well-defined conditions. Our preliminary findings indicate that the oxygen distribution in the microvascular network is heterogeneous and suggest that there is considerable release of oxygen from the arterioles. The time lag of the system for tracking rapid PO2 changes in vitro was shown to be negligible, indicating that dynamic PO2 changes occurring in vivo can also be assessed. This technique should provide a novel tool for the study of oxygen transport and metabolism under normal and abnormal conditions.