In this study we report on the establishment of novel conditions which permit efficient retrovirus-mediated gene transfer of human adenosine deaminase (ADA) into murine hematopoietic progenitors. Using Southern blot analysis and an ADA probe, we demonstrated that prestimulation of bone marrow cells over an in vitro culture of adherent stromal cell layers (ACLs) for two days provides favorable conditions for gene transfer in the absence of exogenous growth factors. In bone marrow transplant recipients reconstituted with retrovirally-marked cells, ADA was detected in spleen, thymus and bone marrow cells of the recipients eight months after transplantation. These observations were also seen in transplants of embryonal hematopoietic stem cells. By using different incubation protocols, it was found that the developmental fate of hematopoietic stem cells varied with the presence of exogenous growth factors or an ACL in the prestimulation phase. Polyclonal hematopoiesis with multiple clones appearing simultaneously was revealed in mice reconstituted with growth factor-stimulated cells four months after transplantation. This was detected by multiple integration patterns of ADA integration into the genomes of individual colony forming units-spleen (CFU-S) in transplantation recipient mice. In contrast, two to five months after transplantation, polyclonal hematopoiesis was not observed in mice reconstituted with cells infected in the absence of growth factors. It appears that utilization of the bone marrow microenvironment through the use of an ACL results in a narrower spectrum of integration patterns, suggesting that a type of oligoclonal or monoclonal hematopoiesis is occurring. These studies demonstrate that an ACL provides novel conditions for successful gene transfer and stable integration of the vector into the genome. Use of an ACL may be advantageous for successful hematopoietic stem cell gene therapy.