A future possibility for treatment of genetic diseases may be gene therapy using autologous cord blood (CB) stem/progenitor cells. This might require cryopreservation of CB stem/progenitor cells prior to purification, gene transduction, and ex vivo expansion of cells. To address this possibility, nonadherent low density T-lymphocyte depleted (NALT-) cells from fresh or cryopreserved cord blood were sorted for CD34 phenotype, transduced with a recombinant retroviral vector encoding Fanconi anemia complementation C (FACC) gene, and cells expanded ex vivo in suspension culture for 7 days with growth factors. The results demonstrate: 1) high recovery of viable cells after thawing; 2) high efficiency purification of CD34 cells from NALT- cells prior to and after cryopreservation; 3) high degree of expansion of nucleated cells and immature progenitors from CD34 cells before and after cryopreservation; 4) efficient transduction with stable integration and expression of newly introduced genes in cryopreserved and then sorted stem/progenitor cells, as detected prior to and after ex vivo expansion; and 5) high efficiency transduction of single isolated CD34 cells obtained from cryopreserved NALT- CB. This information should be of value for future studies evaluating the use of cryopreserved cord blood for gene transfer/gene therapy.