BACKGROUND: Gene therapy is currently being attempted using a number of delivery vehicles including lentiviral-based vectors. The delivery and insertion of a gene using lentiviral-based vectors involves multiple discrete steps, including reverse transcription of viral RNA into DNA, nuclear entry, integration of viral DNA into the host genome and expression of integrated genes. Transduction of murine stem cells by the murine leukemia viruses is inefficient because the expression of the integrated DNA is profoundly blocked. Transduction of human stem cells by lentivirus vectors is also inefficient, but the cause and specific part of the retroviral lifecycle where this block occurs is unknown. RESULTS: Here we demonstrate that the dominant point of restriction of an HIV-1-based lentiviral vector in adult human hematopoietic stem and progenitor cells (HSPCs) from bone marrow and also those obtained following peripheral mobilization is prior to viral DNA integration. We specifically show that restriction of HSPCs to an HIV-1-based lentiviral vector is prior to formation of nuclear DNA forms. CONCLUSIONS: Murine restriction of MLV and human cellular restriction of HIV-1 are fundamentally different. While murine restriction of MLV occurs post integration, human restriction of HIV-1 occurs before integration.