Some cell membranes have been reported to possess rather low permeabilities for CO₂. We have shown previously that the CO₂ permeability (P_CO2) of the membrane of human red cells (RBC) falls from 0.2cm/s to 0.015cm/s upon loss of the functional gas channels Rhesus protein and aquaporin-1. The low intrinsic P_CO2 of some cell membranes is surprising in view of the high P_CO2 values reported for artificial lipid bilayers, 0.4 to 3cm/s, revealing a discrepancy between P_CO2 of lipid bilayers and cell membranes. To investigate this we used the ¹⁸O exchange technique previously employed to analyse P_CO2 of RBC. We produced vesicles 200 nm in diameter using phosphatidycholine - phosphatidylserine (PC-PS, ratio 8:2), which were loaded with high activity of carbonic anhydrase. These vesicles yielded a P_CO2 of 0.13cm/s (37°C). Adding cholesterol (Chol) to give PC:PS:Chol of 8:2:8 resulted in P_CO2 of 0.012cm/s, revealing a strong reduction of P_CO2 by Chol. This latter value is about identical to the P_CO2 of RBC in the absence of gas channels. In another cell model, in tsA201 cells, that express no aquaporin-1, P_CO2 was found to be 0.007cm/s. We conclude that several cell membranes possess rather low CO₂ permeabilities. In contrast to previous views, a major cause appears to be the membrane lipid composition. Cholesterol content has an extremely strong effect, which would agree with the known high Chol content of RBC membrane lipids (50%). It appears that it is expression of gas channels that causes high CO₂ permeabilities in some membranes.