The flow cytometric crossmatch (FCXM) has become an increasingly utilized method to detect low levels of anti-donor antibodies (e.g., anti-HLA) in potential renal allograft recipients. Anti-donor antibodies not apparent in the standard complement-dependent crossmatch, but detectable by the FCXM, are often associated with increased episodes of graft rejection and early graft failure. In this study we examined several parameters of the FCXM in order to establish a standardized methodology. First, we observed that optimal staining results were obtained when the secondary antibody was an Fc-specific F(ab'), anti-human IgG. In contrast to an anti-whole immunoglobulin antibody, the anti-Fc specific reagent did not react with surface immunoglobulin on B cells but was reactive with cytophilic immunoglobulin present on CD16+ cells. Next we determined that dualcolor analysis was superior to single-color analysis both for the evaluation of T cell reactivities and for the discrimination of T cell from B cell reactivities. Additionally, dual-color analysis revealed that the density of class I histocompatibility antigens on B cells is greater than on T cells, indicating that B cells may be a more sensitive target for detecting low levels of anti-class I antibodies. Finally, we determined that a shift in the mean fluorescence intensity of greater than 10 channels on a 256-channel, 3-decade log scale was indicative of a positive FCXM. The data presented in these studies provide the basis for performing standardized dual-color FCXM with increased sensitivity and specificity.