Adenoviral vector-mediated transient gene expression can provide new possibilities for ex vivo manipulation of quiescent hematopoietic stem cells (HSC). In order to define a suitable expression cassette for high levels of transgene expression in HSCs, we have studied the level of transgene expression in human CD34+CD38- cells using adenoviral vectors with various gene expression cassettes encoding the enhanced green fluorescence protein (EGFP) gene. CD34+ hematopoietic cells were cultured in serum-free medium with megakaryocyte growth and development factor (MGDF) alone for supporting the survival of primitive progenitors or with MGDF, c-kit ligand (KL) and flt3 ligand (FL) for inducing proliferation of primitive progenitors. With all the vectors tested, higher percentages of EGFP expressing cells were found in CD34+CD38- cells than those in CD34+CD38high cells from all donors tested. The phosphoglycerate kinase (PGK)-1 promoter was found to allow higher levels of EGFP expression than the human cytomegalovirus (HCMV) promoter in CD34+CD38- cells. Replacing the SV40 polyadenylation signal with the human beta-globin gene IVS2 and polyadenylation signal in the expression cassette (Ad5xPGK-EGFP-beta-globin) enhanced the level of EGFP expression markedly further. These results provide a guideline for the development of adenoviral vectors for gene expression in human primitive hematopoietic progenitor cells. Gene Therapy (2000) 7, 2132-2138.