Gene transfer or gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. Interferon-alpha (IFN-alpha) has been used in the management of leukemia but its diverse adverse activities with multiple potential side effects, possibly unrelated to therapeutic targets, may negatively influence the ability of IFN-alpha to treat this disorder. Therefore, we examined the ability of adenovirus (Ad)-IFN-alpha gene construct to transfect normal (CD34+ cells) and chronic myelogenous leukemia (CML) bone marrow mononuclear cells (BMMNC) and the transient overexpression of IFN-alpha in these cells. Ad-cytomegalovirus promoter driven IFN-alpha (AdCMV-IFN-alpha) at multiple doses was assessed to transfect highly purified CD34+ cells in liquid culture, and optimal transduction of CD34+ cells was achieved using 120 plaque forming units. Flow cytometric determinations revealed that there was no significant difference in cell viability for the 4 h or 24 h transfection periods. Immunoassay of IFN-alpha produced by CD34+ cells shows that IFN-alpha levels increased several fold in transfected cells. Transient expression of the IFN-alpha gene did not suppress proliferation of CD34+ progenitors as indicated by BFU-E or colony forming units-granulocyte-macrophage (CFU-GM) growth. Reverse transcriptase/polymerase chain reaction analysis of RNA from CD34+ harvested CFU-GM progenitor cells demonstrated transient IFN-alpha mRNA expression. Similarly, CML BMMNC were transfected with AdCMV-IFN-alpha under similar conditions as described for CD34+ cells. BMMNC cells exposed to adenovirus for 24 h and 48 h were found to express IFN-alpha at a substantial level. This in vitro data suggest that Ad-mediated gene transfer of IFN-alpha into hematopoietic stem cells can be achieved and that the IFN-alpha gene can be translated into its specific mRNA in CD34 progenitor cells.