Abstract The methods available for separating mixed populations of red blood cells (RBCs) are not completely satisfactory. The purpose of this study was to evaluate the utility of microbead columns for the separation of mixtures of RBCs of different blood groups. Suspensions of RBCs positive for nine different blood group antigens were mixed with RBCs lacking the antigen so that 1%, 5%, 10%, 15%, or 25% of the antigen-positive RBCs were represented. After agglutination by the appropriate antiserum, the antigen-positive RBCs were separated from the antigen-negative RBCs by using microbead columns. The average recovery of the antigen-negative RBCs in the effluent of the columns of the 135 mixtures of RBCs tested was 83% ± 5% (mean ± SD). The absence of contaminating antigen-positive RBCs was established serologically and by flow cytometry. The procedure was effective in removing as little as 1% of antigen-positive cells from a mixture. Microbead columns offer a simple and efficient method for separating mixtures of RBCs for biochemical, clinical, and serologic studies.