We present a fast, simple, and accurate method to determine the affinity constants of antibodies that bind to cell surface antigens. This procedure utilizes intact cells and native, unmodified antibody in a conventional enzyme-linked immunosorbent assay. Target cells are incubated with serial dilutions of antibody and allowed to reach equilibrium. Cells are then pelleted by centrifugation, and aliquots of unbound antibody in the supernatant are added to a microtiter plate precoated with capture antibody and measured in a conventional enzyme-linked immunosorbent assay (ELISA). We measured the affinity constant of murine monoclonal antibody CLB-1H-gran2, which binds to K562 cells (a human erythroleukemia line), and compared the ELISA-based results to those obtained by flow cytometric determination of antibody affinity. The affinity constants obtained by the two methods are in good agreement. The affinity constant is calculated utilizing only the concentrations of bound and free antibody, so that the actual antigen concentration (or number of antigenic sites per cell) need not be known. However, the number of antibody molecules bound per cell can be estimated from the results.