It has recently been reported that the polycation Polybrene (hexadimethrine bromide), like ristocetin, agglutinates platelets more extensively in the presence of normal plasma than von Willebrand plasma. Since we have previously proposed that ristocetin may initiate agglutination by reducing platelet surface charge, I investigated the correlation between Polybrene's ability to induce agglutination and alter platelet electrophoretic mobility. In the absence of plasma, low concentrations of Polybrene produced small platelet aggregates and reduced the electrophoretic mobility. Higher concentrations were needed to produce small platelet aggregates in the presence of von Willebrand plasma; these same concentrations produced more rapid agglutination and much larger aggregates in the presence of normal plasma. The reductions in electrophoretic mobility were also greater in the presence of normal than von Willebrand plasma. Both agglutination and the reduction in mobility could be partially reversed with citrate. Vancomycin, an antibiotic that inhibits ristocetin-induced agglutination with some specificity, inhibited both Polybrene-induced agglutination and the reductions in platelet mobility. These data are consistent with our electrostatic model and support the view that reductions of platelet surface charge may be necessary (but perhaps not sufficient) to initiate the interaction between platelets and von Willebrand factor.