The Wrb antigen is a high-frequency human erythrocyte antigen invariably absent from En (a-) erythrocytes, which lack glycophorin A. However, glycophorin A from En (a+) Wr (a+b-) red cells has an amino acid sequence identical to that of glycophorin A from Wr (b+) erythrocytes. Evidence has suggested that the Wrb antigen may require the interaction of glycophorin A with either a lipid moiety or with another erythrocyte-integral membrane protein, band 3. We have investigated the role of band 3 in Wrb expression using murine monoclonal antibodies (MoAbs) with Wrb specificity. These antibodies reacted by radioimmunoassay (RIA) only with cells expressing both glycophorin A and band 3. In immunoprecipitation studies, Wrb antibodies immunoprecipitated both band 3 and glycophorin A, while antibodies specific for band 3 or glycophorin precipitated only the protein with which they were reactive. These data strongly suggest that band 3 is the other membrane component necessary for expression of Wrb and that band 3 and glycophorin A are closely associated in the erythrocyte membrane.