Using the mass spectrometric ¹⁸O exchange technique we show that the membranes of several cells with a normal cholesterol content of ~40 mol% have a low intrinsic permeability for CO₂ (P_CO2[asymp]0.01 cm/s). We also show that artificial phospholipid vesicles, when enriched with cholesterol to 40 mol%, possess about the same low P_CO2, which is > 2 orders of magnitude lower than P_CO2 of pure phospholipid vesicles. The suggestion from these observations is that cholesterol highly effectively reduces P_CO2 of membranes. This hypothesis is tested here by using methyl-ß-cyclodextrin, either free of or preloaded with cholesterol, to either drastically reduce or increase the cholesterol content of the membranes of intact MDCK cells. From a control P_CO2 of 0.017 cm/s, reduction of membrane cholesterol causes an increase in P_CO2 to > 0.75 cm/s, and increasing cholesterol leads to a fall in P_CO2 to 0.0065 cm/s. Thus, the ~40 mol% cholesterol content exhibited by most cell membranes appears to confer a rather low P_CO2 on them. We cite literature data that show that a membrane cholesterol content of 75 mol% produces an extremely low P_CO2 of 0.001 cm/s (e.g. in the apical membranes of some epithelia), 10x lower than P_CO2 of MDCK cells. By expressing aquaporin-1 in MDCK cells, we show that on the background of their moderately low P_CO2 of 0.017 cm/s, the gas channel aquaporin-1 causes a significant increase in the conductivity of the plasma membrane for CO₂. We conclude that many cell membranes possess a rather low intrinsic CO₂ permeability, making it necessary to insert protein gas channels to make them highly CO₂-permeable.