A B $ T R A C T Diffusion of 14C-labeled CO2 was measured through lipid bilayer membranes composed of egg lecithin and cholesterol (1:1 mol ratio) dissolved in ndecane. The results indicate that CO2, but not HCOa-, crosses the membrane and that different steps in the transport process are rate limiting under different conditions. In one series of experiments we studied one-way fluxes between identical solutions at constant pCO2 but differing [HCOa-] and pH. In the absence of carbonic anhydrase (CA) the diffusion of CO2 through the aqueous unstirred layers is rate limiting because the uncatalyzed hydration-dehydration of COo is too slow to permit the high [HCOa-] to facilitate tracer diffusion through the unstirred layers. Addition of CA (ca. 1 mg/ml) to both bathing solutions causes a 10-100-fold stimulation of the COo flux, which is proportional to [HCOa-] over the pH range 7-8. In the presence of CA the hydration-dehydration reaction is so fast that COs transport across the entire system is rate limited by diffusion of HCOs-through the unstirred layers. However, in the presence of CA when the ratio [HCOs-+ COa=]:[CO0] > 1,000 (pH 9-10), the COo flux reaches a maximum value. Under these conditions the diffusion of CO~ through the membrane becomes rate limiting, which allows us to estimate a permeability coefficient of the membrane to COo of 0.35 cm s -1. In a second series of experiments we studied the effects of CA and buffer concentration on the net flux of CO~. CA stimulates the net COo flux in well buffered, but not in unbuffered, solutions. The buffer provides a proton source on the upstream side of the membrane and proton sink on the downstream side, thus allowing HCOs-to facilitate the net transport of COo through the unstirred layers.