Recent studies have strongly suggested that aquaporin-1 (AQP1) and the HCO3-/Cl- transporter (AE1) may function as gas pores thus aiding CO2 transport across biological membranes. The hypothesis both proteins may serve as gas transfer routes within one membrane was tested and confirmed on CO2 transport in RBC ghosts loaded with the proton sensitive dye BCECF which monitored intracellular acidification as a measure for CO2 entry: Any specific acidification was restricted to the intracellular space, suppressed by functional inhibitors of AQP1 and AE1 (HgCl2 & DIDS), and required the catalytic activity of CA as shown with ETX, a membrane permeable inhibitor of CA. We investigated the reversibility of acidification, which plays a role in regulatory mechanisms like pH and volume homeostasis. Ghosts suspensions were incubated for 10 min prior to the onset of CO2. After 60 s ETX (10 mM) was injected into the cuvette and CO 2 was replaced with N2; the rate of pH recovery was about .25 of control. In a second experimental series ETX was injected, while the gas supply stayed the same; the rate of transient pH recovery was about 4-fold compared to control. Our results indicate that acidification of RBC ghosts is reversible but needs the catalytic activity of CA for molecular CO2 to exit the cell.