The carbohydrate-binding specificities of pokeweed mitogen (PWM) were studied by interaction between '*'I-PWM and purified glycoproteins. Human erythrocyte-membrane, band-3 glycoprotein bound l'sl-PWM, but other glycoproteins containing triple-branched (tri-antennary)-complex-, double-branched (biantennary)-complex-, hybrid-, high-mannose, or small-mucin-type carbohydrate chains failed to bind '*'I-PWM. Pretreatment of human erythrocytes with endo@ D-galactosidase prevented '"'I-PWM binding to the erythrocytes, as well as to band-3 glycoprotein from these cells. Poly(N-acetyllactosamine) glycopeptides and complex-type glycopeptides purified from band-3 glycoprotein and porcine thyroglobulin, respectively, were labeled at their nonreducing, terminal D-galactosyl or 2-acetamido-2-deoxy-D-galactosyl groups by D-galactose oxidase-sodium borotritide treatment and examined for interaction with agarose-immobilized PWM isolectins. From the binding behavior of these labeled glycopeptides on columns of immobilized-PWM isolectins, and from the interaction of various glycoproteins bearing known carbohydrate chains with "SI-PWM, it was concluded that all three major PWM isolectins (Pa-l, Pa-2, and Pa-4) bind specifically to poly(l\r-acetyllactosamine) structures.