CO₂ permeability, P_CO2, was measured by ¹⁸O exchange in human normal and aquaporin 1-deficient (AQP1-deficient (1)) red cells and in Xenopus laevis oocytes expressing human AQP1. In normal red cells we find a P_CO2 of 0.28 ± 0.21 cm/s (mean ± S.D.; n=70), which is 3-4 orders of magnitude greater than the permeability of this membrane for HCO₃^– (P_HCO3-= 1.5×10^-3 cm/s). In the presence of 1 mM of the mercurial pCMBS, P_CO2 of red cells from normal individuals falls significantly (P<0.05) to 0.07 cm/s (i.e., 25% of normal). In AQP1-deficient red cells from Colton-null individuals, P_CO2 is also ~25% (P<0.05) of the uninhibited value in red cells from normal individuals. In the AQP1-null cells, pCMBS has no significant effect on P_CO2, indicating that the target molecule of pCMBS is indeed AQP1. These results suggest that AQP1 in red cells is the major pathway for molecular CO₂. This view is confirmed by the finding that P_CO2 of Xenopus laevis oocytes approximately doubles with expression of human AQP1 (P<0.04). In red cells from normal individuals, 100 μM DIDS reduces P_CO2 from 0.25 cm/s to 0.05 cm/s (P<0.02). In AQP1-null red cells, DIDS reduces P_CO2 from 0.07 to 0.012 cm/s (P<0.05). This last value may represent the basal P_CO2 of the membrane due to the lipid phase alone. We conclude that, in contrast to classical views, transport of molecular CO₂ across red-cell membranes occurs mainly via proteins.