We have determined CO₂ permeabilities, P_CO2, of red cells of normal human blood and of blood deficient in various blood group proteins by a previously described mass spectrometric technique. While P_CO2 of normal red cells is 0.15 cm/s, we find in red blood cells (RBCs) lacking the Rh protein complex (Rh_null) a significantly reduced P_CO2 of 0.07 cm/s ±0.02 cm/s (P<0.02). This value is similar to the value we have reported previously for RBCs lacking aquaporin-1 protein (AQP-1_null), suggesting that each of the Rh and AQP-1 proteins is responsible for 1/2 of the normal CO₂ permeability of the RBC membrane. Four other blood group deficiencies tested lack diverse membrane proteins but exhibit normal CO₂ permeability. The CO₂ pathway constituted by Rh proteins was inhibitable at pH_e= 7.4 by NH₄Cl with an I₅₀ of 10 mM corresponding to an I₅₀ for NH₃ of 0.3 mM. The pathway independent of Rh proteins, presumably that constituted by AQP-1, was not inhibitable by NH₄Cl/NH₃. However, both pathways were strongly inhibited by DIDS, which accounts for the marked inhibitory effect of DIDS on normal P_CO2, while in contrast another AE1 inhibitor, DiBAC, does not inhibit P_CO2, although it markedly reduces P_HCO3-. We conclude that Rh protein, presumably the Rh-associated glycoprotein RhAG, possesses a gas channel that allows passage of CO₂ in addition to NH₃.—Endeward, V., Cartron, J.-P., Ripoche, P., and Gros, G. RhAG protein of the Rhesus complex is a CO₂ channel in the human red cell membrane.