Retroviral gene transfer of human cytidine deaminase into murine hematopoietic cells has been shown to confer drug resistance against cytidine analogues such as araC (1-β-D-arabinofuranosylcytosine) and gemcitabine (2′,2′-difluorodeoxycytidine). Since data for primary human hematopoietic cells are lacking so far, we transduced human CD34+ enriched umbilical cord blood cells with the retroviral vector SF91-CDD-IRES-EGFP containing the cDNA of cytidine deaminase (CDD) and EGFP as a reporter gene. The transduced cells were tested in clonogenic assays for resistance against increasing concentrations of araC or gemcitabine. Gene transfer of SF91-CDD-IRES-EGFP resulted in a transduction efficiency of 28 ± 2% (n = 4). A significant higher survival of CFU-C in the presence of araC could be demonstrated as compared with the mock control expressing EGFP only. This protective effect was improved by FACS enrichment of EGFP+ cells prior to functional analysis, and was observed for a dose range of 30 – 500 nM araC. Gemcitabine resistance was also tested with cells enriched for EGFP expression. For CFU-C a significant higher survival of colonies at concentrations of 8 and 10nM could be shown. This protection was more pronounced for CFU-GM than for BFU-E. The IC50 value increased 3fold for CFU-GM but only 1.4fold for BFU-E and 1.8fold for total CFU-C in comparison to the mock control (n = 4). In vitro selection of CDD transduced progenitor cells was investigated by incubation in the presence of 30 – 100nM araC for 4 or 8 days. Before and after culture cells were analysed for EGFP expression and tested in clonogenic assays for araC resistance. Significant selection of CDD transduced cells as measured by EGFP expression was observed for all examined concentrations. After 4 days incubation with 60nM araC clonogenic assays revealed a significant selection of progenitor cells resistant to 100 – 500nM araC. Incubation of transduced cells in 6 – 15nM gemcitabine for 4 or 8 days likewise allowed successful in vitro selection. Transplantation of CDD transduced CD34+ cells in sublethally irradiated NOD/SCID mice resulted in up to 81% engraftment of human CD45+ cells and a rate of up to 4.6% of human CD45+/EGFP+ cells after 8 weeks. These data demonstrate that retroviral gene transfer of CDD protects human hematopoietic cells from the cytotoxic drugs araC and gemcitabine and allows selection of transduced cells. Successful engraftment of CDD transduced cells in NOD/SCID repopulating cells could be observed.