BACKGROUND The lymphocytes separated from whole blood are used in HLA flow cytometry crossmatch (FCXM) for renal transplantation. In this study, the methodology of whole blood flow cytometry was applied to FCXM, omitting lymphocyte separation step. METHODS In the 20 cases (including positive 5 cases) of T cell FCXM for renal transplantation, the standard assay using the separated mononuclear cells (MNC) was compared with the two variant assays using whole blood. In the latter assay, the donor whole blood was incubated with the excessive recipient serum. The red cells were lysed (lysed whole blood, LWB). Otherwise, instead of red cell lysis, the signals of T cells among whole blood (WB) were acquired using fluorescence triggering. The sample/negative control mean fluorescence intensity (MFI) ratio was calculated for the interpretation. RESULTS The MFI ratio of the 20 cases by MNC, LWB and WB assay were 4.9+/-8.1, 5.4+/-9.7 and 4.8+/-7.8, respectively. Both LWB and WB assay were not significantly different from MNC assay (P= 0.313, 0.831, respectively, paired t-test). The qualitative determinations were concordant in all cases, except for one case which was weakly positive with MFI ratio 2.2 by LWB assay. CONCLUSIONS The assays using whole blood were comparable to the standard assay in FCXM for renal transplantation. This study indirectly supports that the variant methods can be used reliably in the case of the MNC preparation erroneously mixed with other blood cells.