Abstract: Objective To compare the characteristics of three-and two-color flow cytometry for immunologic surveillance of toll-like receptor 4(TLR-4) after renal transplantation.Method The expressions of CD14,TLR4 and CD80 in peripheral blood monocytes were measured in 10 patients after renal transplantation by three-and two-color fluorescent staining flow cytometry,respectively.The percentages of TLR4 and CD80 in CD14-positive monocytes were calculated,and the characteristics of two methods of flow cytometry were compared.Results The two detection methods did not significantly differ in their detection of TLR4(P=0.198) and CD80(P=0.872) expressions in CD14-positive monocytes,and the measurements of the two methods for the same sample showed positive correlation(r=1,P=0.000 for TLR4 and r=0.999,P=0.000 for CD80).Three-color flow cytometry was also capable of detecting monocytes positive for both TLR4 and CD80.Compared with two-color flow cytometry,three-color flow cytometry provided more information and required less blood sample and monoclonal antibody.Conclusions Three-and two-color flow cytometry have almost the same accuracy in immunologic surveillance of TLR4 after renal transplantation.The former method also provides information of the simultaneous expression of TLR4 and its down stream molecule CD80,which could be critical for imploring the mechanisms by which TLR4 takes part in alloimmunity through modulating CD80 expression,and requires less blood sample and monoclonal antibody.