Abstract The trachea, pulmonary artery, and left atrium were cannulated, and the isolated lungs floated, trachea oriented downward, on a layer of paraffin oil in an incubated chamber at 37 °C. With the trachea partially filled with fluid to establish electrical continuity, the electrical potential of the tracheal fluid, and of the pleural surface were measured with respect to the fluid in the vascular bed. With phosphate-buffered Ringer solution in the trachea, in the vascular bed and on the pleural surface, the tracheal vascular potential, and the vascular-pleural potential, were found to be on the order of — 12 and — 5 mV, respectively. These potentials were greatly reduced or abolished by low temperature, lack of oxygen, cyanide, and ouabain, suggesting that they were generated by an energy-dependent transport of ions. When isotonic sodium chloride solution was used in place of Ringer solution, potentials similar to the above were obtained. However, when choline was substituted for sodium, or sulfate was substituted for chloride, the vascular-pleural potential was abolished. This suggests that both sodium and chloride ion are necessary for the genesis of the vascular-pleural potential. In the intact rat the tracheal-vascular potential (measured between the inside of the trachea and the blood in the carotid artery) was on the order of — 7 mV; with the chest open the vascular-pleural potential (measured between the blood in the carotid artery and the pleural surface of the lungs) was on the order of -4mV.