Objective: It has been reported that intracellular carbonic anhydrase (CA) II binds to the C-terminal domain of AE1 and suggested that this increases the transport rate of AE1. The goal of our study was a) to detect the intracellular distribution and possible co-localization of CAII and AE1 in living cells, b) to measure bicarbonate transport by AE1 in the absence and presence of CAII in tsA201 cells and human red blood cells (RBCs), and c) to apply a mathematical simulation to the question whether close proximity of AE1 and CA activity at the membrane can be expected to enhance HCO3- and CO2 transport. Methods: We expressed in tsA cells fusion proteins of AE1 with yellow fluorescent protein and of CAII with cyan fluorescent protein, and determined the distribution of these proteins in tsA cells by confocal laser scanning microscopy (CLSM). We measured membrane HCO3- permeabilities (PHCO3-) of tsA cells and of CAII-deficient RBCs by mass spectrometry using 18O-labelled HCO3-. Results: a) CLSM shows that AE1 expressed in tsA cells is exclusively associated with the plasma membrane, while CAII is homogeneously distributed in the cytoplasm. There is no enrichment of CAII at the plasma membrane. PHCO3- of tsA cells exhibits a marked increase upon expression of AE1. Co-expression of CAII with AE1 does not lead to a further increase in PHCO3-. b) We find no difference in PHCO3- of normal and CAII-deficient human RBCs. c) In a mathematical simulation we show that a cytoplasmic CA dramatically improves bicarbonate transport by AE1. An equivalent activity of CA exclusively attached to the internal side of the membrane is slightly less effective. Conclusions: 1) There is no co-localization of AE1 and CAII at the membrane upon co-expression in tsA cells; 2) PHCO3- is independent of the presence of CAII in tsA cells as well as in RBCs; 3) Activity of CA close to the AE1 transporters has no advantage for HCO3- transfer over a CA distributed homogeneously in the cytoplasm.