We tested for the presence of CO2-sensitive venous chemoreceptors in anesthetized, paralyzed, artificially ventilated cats (N = 8). Systemic venous PCO2 was elevated (venous CO2 loading) by continuously passing blood withdrawn from the femoral artery (20 ml/min) through an extracorporeal gas exchanger, ventilated with 50% CO2 and 50% O2, and returning this hypercapnic blood to the femoral vein. Respiratory output was assessed by means of the amplitude of the integrated phrenic neurogram. Results of venous CO2 loading were compared to those of airway CO2 loading in which inspired CO2 levels were adjusted to give the same arterial PCO2 (PaCO2) as in venous loading. Despite large differences in mixed venous PCO2 (PvCO2) during venous CO2 loading (PvCO2 = 55 Torr, PaCO2 = 37 Torr) compared to airway CO2 loading (PvCO2 = 45 Torr, PaCO2 = 37 Torr), phrenic output was unchanged. However, phrenic output was elevated 33% when PaCO2 was increased 6-7 Torr by raising inspired CO2 and reduced 50% when PaCO2 was lowered 6-7 Torr by lowering inspired CO2, thereby substantiating the responsiveness of the respiratory control system to changes in PaCO2. The respiratory output response to changes in venous CO2 was also tested at a higher PaCO2 (40 Torr, created by adding 1% CO2 to the inspired air) and, as before, no change in phrenic output occurred when PvCO2 was elevated at a constant PaCO2. These experiments provide direct evidence for the absence of chemoreceptors in the central veins, right heart, and pulmonary arterial system of the cat that would respond to changes in PvCO2.