Unstirred layers around cells can be a problem when membrane permeabilities are to be measured. Especially in the case of very high gas permeabilities, the diffusion resistance of extracellular unstirred layers greatly contributes to the apparent membrane resistance and can make measurements of gas permeabilities impossible. We have developed a new method to determine the effect of unstirred layers on red cell CO₂ permeability (P_CO2). We use the mass spectrometric technique to measure P_CO2 of red cell membranes. The unstirred layer effect in this experimental setup was determined by studying the influence of the solution viscosity (h) on the thickness of unstirred layers around cells. Hydrodynamic theory predicts that the thickness d of an unstirred layer increases with increasing h and with h = 0 unstirred layers disappear. We measured P_CO2 in saline and added dextran to establish different dextran concentrations between 0% and 10%. With 10% dextran the apparent P_CO2 falls to 1/3 of the P_CO2 in saline, which indicates the increased thickness of the unstirred layer. Plotting 1/P_CO2 vs. h gives a straight line whose extrapolation to h = 0 gives the P_CO2,corr = 0.16 cm/s that is expected after correction for the extracellular unstirred layer. This permeability is ~30% higher than the value of 0.12 cm/s measured in saline. From this, we calculate a thickness of the extracellular unstirred layer of d = 0.5mm in the saline measurements, using the equation d = (1/P_{CO2, apparent}– 1/_{PCO2, corr}) · D_{CO2, solution}, where D_{CO2, solution} is the CO₂ diffusion coefficient of in saline. We conclude that in well-stirred solutions unstirred layers around red cells are in the range of 0.5 mm, which leads to a moderate underestimation of the membrane CO₂ permeability by 30%. In addition, we developed a theoretical model to determine the effect of an intracellular unstirred layer on P_CO2. With this model we find that an intracellular unstirred layer reduces P_CO2 by another 30%, and considering both intra– and extracellular unstirred layers, we estimate a true membrane permeability of 0.2 cm/s.