We examined the dependence of respiratory rhythm generation on the rate of CO2 production (VCO2) in six awake sheep using a venous-to-venous extracorporeal perfusion circuit that included two carbon dioxide membrane lungs (CDML) designed to facilitate the removal of CO2 from blood. As progressively greater proportions of the resting VCO2, of the sheep were removed from the venous blood by the CDML, there were proportional reductions in pulmonary CO2 excretion and in minute volume of ventilation (VE); when CO2 was removed by the CDML at a rate equal to its metabolic production by the sheep, VE fell to zero, with normal values of arterial PCO2, pH, and PO2. The results indicate that effective respiratory rhythm generation is critically dependent on stimuli related to VCO2.