We have reinvestigated the evidence for physical association of and direct functional interaction between carbonic anhydrase II (CAII) and the anion exchanger 1 (AE1), called the "metabolon" hypothesis. In tsA201 cells co-expressing heterologous fluorescent fusion proteins CAII-CyPet and YPet-AE1, YPet-AE1 is clearly associated with the cell membrane, whereas CAII-CyPet is homogeneously distributed throughout the cell in a cytoplasmic pattern. FRET measurements fail to detect close proximity of YPet-AE1 and CAII-CyPet. Both the CAII and 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 DIDS. Expression of the non-catalytic CAII mutant V143Y leads to a drastic reduction of endogenous CAII and to a corresponding reduction of 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 wildtype CAII. These variously co-transfected tsA201 cells exhibit a simple linear relationship between cellular P_HCO3- and intracellular CA activity. The relationship is consistent with changes in HCO₃^- transport that reflect either substrate supply to or removal from AE1 via altered cytoplasmic CA activity, without requirement for a hypothesized CAII-AE1 metabolon involving physical interaction. A functional contribution of the hypothesized CAII-AE1 metabolon to erythroid AE1-mediated HCO₃^- transport was 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, again providing no support for the proposed functional importance of a physical interaction of CAII and AE1.