The antigenic determinants of the blood group P family (P1, P, Pk, and LKE antigens) are chemically based on Gal alpha 4Gal. For human erythrocytes it has been claimed that the majority of P1 determinants are expressed in glycoproteins, mainly band 4.5 (Haselberger, C. G., and Schenkel-Brunner, H. (1982) FEBS Lett. 149, 126-128). In the present work, the existence of Gal alpha 4Gal in glycoproteins of erythrocyte membranes (ghosts) of P1 positive and negative human individuals was carefully analyzed on replicas of sodium dodecyl sulfate-polyacrylamide electrophoresis gels using specific reagents (Escherichia coli HB101/pDC1 expressing the Pap gene and monoclonal antibodies with specificities for P1 and Pk antigens). No binding to glycoproteins was detected with any of these ligands when the ghosts had been pretreated with butanol to remove glycolipids. Therefore, all antigenic determinants of the P blood group family on human red cells are exclusively expressed in glycolipids and are absent from glycoproteins.