BACKGROUND: CD34 positive cell enumeration by flow cytometry is currently used to determine the optimal timing of peripheral blood stem cell collections (PBSC) and to predict engraftment of stem cell transplantation. However, the technical problems and lack of a standardized method are sources of significant variability in the quantitation of the CD34 positive cells. ProCOUNT(TM) (Beckon Dickinson Immuno- cytometry System, USA) kit for three color flow cytometric analysis was introduced to enumerate CD34 positive cells using a standardized method. This study was conducted to evaluate the usefulness of the three color method, ProCOUNT(TM), in comparison with two color method. METHODS: CD34 positive cells from 25 cord blood samples were enumerated by two methods, two color (CD34-PE/CD45-FITC) and three color (ProCOUNT(TM) , nucleic acid dye/CD34-PE/ CD45-PerCP) flow cytometric analysis, in which CD34 positive cells were counted directly in comparison with counting beads introduced in the sample. RESULTS: The count of CD34 positive cells in the cord blood was 28.3(+/-20.0)/uL and 20.9 (+/-16.0) /uL by three color and two color methods, respectively, The number of CD34 positive cells enumerated by ProCOUNTTM kit was well correlated with that by two color method, but the count was significantly higher in the former method (p<0.01). CONCLUSIONS: In the three color method, loss of stem cells was significantly lower than that in the two color method, and it was possible to obtain a direct count of CD34 positive cells by using a standardized procedure.