Vancomycin, an antibiotic similar in structure to ristocetin, is used to treat staphylococcal infections. However, vancomycin-induced hemagglutination complicated red cell (RBC) serologic testing in the blood bank. At concentrations greater than 3.0 mg per ml, vancomycin caused spontaneous macroscopic red cell (RBC) aggregation; concentrations of 2.0 and 2.5 mg per ml were associated with weakly positive aggregation with anti-IgG and polyspecific antiglobulin reagents negative with anti-complement; and concentrations less than 1.5 mg per ml had no apparent effect. Ficin-treated RBCs demonstrated negative reactions with the antiglobulin reagents. Vancomycin-induced aggregation was reversed partially with 0.2 M trisodium citrate, and supernatant transfer studies showed that normal RBCs retained a significantly (p less than 0.025) greater percentage of vancomycin than did ficin-treated RBCs. Vancomycin causes the aggregation of RBCs, which can be a source of confusion in the blood bank. The mechanism(s) through which vancomycin enhances aggregation may be related to its polycationic properties and to its direct protein binding to the RBC membrane, although other nonimmunologic mechanisms may be operative.