The resting membrane potential of the human erythrocyte is largely determined by a constitutive Cl(-) conductance approximately 100-fold greater than the resting cation conductance. The 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive electroneutral Cl(-) transport mediated by the human erythroid Cl(-)/HCO(3)(-) exchanger, AE1 (SLC4A1, band 3) is >10,000-fold greater than can be accounted for by the Cl(-) conductance of the red cell. The molecular identities of conductive anion pathways across the red cell membrane remain poorly defined. We have examined red cell Cl(-) conductance in the Ae1(-/-) mouse as a genetic test of the hypothesis that Ae1 mediates DIDS-sensitive Cl(-) conductance in mouse red cells. We report here that wildtype mouse red cell membrane potential resembles that of human red cells in the predominance of its Cl(-) conductance. We show with four technical approaches that the DIDS-sensitive component of erythroid Cl(-) conductance is reduced or absent from Ae1(-/-) red cells. These results are consistent with the hypothesis that the Ae1 anion exchanger polypeptide can operate infrequently in a conductive mode. However, the fragile red cell membrane of the Ae1(-/-) mouse red cell exhibits reduced abundance or loss of multiple polypeptides. Thus, loss of one or more distinct, DIDS-sensitive anion channel polypeptide(s) from the Ae1(-/-) red cell membrane cannot be ruled out as an explanation for the reduced DIDS-sensitive anion conductance.