To investigate the differentiation of pre-preparation method to determine CD4 on T lymphocyte in blood, CD4 expression on T lymphocyte of monkey was determined by the flow cytometrey as a quantitative analysis with three different pre-preparation methods. These results showed that (b) method (whole blood was labeled by FITC-CD4 McAb. Then red blood cells were dissolved by erythrocytolysis) was better (a) method (red blood cells were dissolved by erythrocytolysis in whole blood. Then it was labeled by FITC-CD4 McAb). CD4 results with (b) method were equal to (c) method (traditional method of lymphocyte separated). But the volume of sample by (b) method only was 1/5 of (c) method. The pre-preparation of (b) method also was sampler than (c) method.Morphologic study by laser scanning confocal microscopy showed (b) method was similar to (c) method. Both of them had a clear fluorescent labeled on the cell membrane but not in the cell.