We have investigated the previously published 'metabolon hypothesis' postulating that a close association of the anion exchanger 1 (AE1) and cytosolic carbonic anhydrase II (CAII) exists that greatly increases the transport activity of AE1. We study whether there is a physical association of and direct functional interaction between CAII and AE1 in the native human red cell and in tsA201 cells coexpressing heterologous fluorescent fusion proteins CAII-CyPet and YPet-AE1. In these doubly transfected tsA201 cells, YPet-AE1 is clearly associated with the cell membrane, whereas CAII-CyPet is homogeneously distributed throughout the cell in a cytoplasmic pattern. Förster resonance energy transfer measurements fail to detect close proximity of YPet-AE1 and CAII-CyPet. The absence of an association of AE1 and CAII is supported by immunoprecipitation experiments using Flag-antibody against Flag-tagged AE1 expressed in tsA201 cells, which does not co-precipitate native CAII but co-precipitates coexpressed ankyrin. Both the CAII and the AE1 fusion proteins are fully functional in tsA201 cells as judged by CA activity and by cellular HCO3(-) permeability (P(HCO3(-))) sensitive to inhibition by 4,4-Diisothiocyano-2,2-stilbenedisulfonic acid. Expression of the non-catalytic CAII mutant V143Y leads to a drastic reduction of endogenous CAII and to a corresponding reduction of total intracellular CA activity. Overexpression of an N-terminally truncated CAII lacking the proposed site of interaction with the C-terminal cytoplasmic tail of AE1 substantially increases intracellular CA activity, as does overexpression of wild-type CAII. These variously co-transfected tsA201 cells exhibit a positive correlation between cellular P(HCO3(-)) and intracellular CA activity. The relationship reflects that expected from changes in cytoplasmic CA activity improving substrate supply to or removal from AE1, without requirement for a CAII-AE1 metabolon involving physical interaction. A functional contribution of the hypothesized CAII-AE1 metabolon to erythroid AE1-mediated HCO3(-) transport was further tested in normal red cells and red cells from CAII-deficient patients that retain substantial CA activity associated with the erythroid CAI protein lacking the proposed AE1-binding sequence. Erythroid P(HCO3(-)) was indistinguishable in these two cell types, providing no support for the proposed functional importance of the physical interaction of CAII and AE1. A theoretical model predicts that homogeneous cytoplasmic distribution of CAII is more favourable for cellular transport of HCO3(-) and CO2 than is association of CAII with the cytoplasmic surface of the plasma membrane. This is due to the fact that the relatively slow intracellular transport of H(+) makes it most efficient to place the CA in the vicinity of the haemoglobin molecules, which are homogeneously distributed over the cytoplasm.