We have probed the nucleoprotein organization of Moloney murine leukemia virus (MLV) pre-integration complexes using a novel footprinting technique that utilizes a simplified in vitro phage Mu transposition system. We find that several hundred base pairs at each end of the viral DNA are organized in a large nucleoprotein complex, which we call the intasome. This structure is not formed when pre-integration complexes are made by infecting cells with integrase-minus virus, demonstrating a requirement for integrase. In contrast, footprinting of internal regions of the viral DNA did not reveal significant differences between pre-integration complexes with and without integrase. Treatment with high salt disrupts the intasome in parallel with loss of intermolecular integration activity. We show that a cellular factor is required for reconstitution of the intasome. Finally, we demonstrate that DNA-protein interactions involving extensive regions at the ends of the viral DNA are functionally important for retroviral DNA integration activity. Current in vitro integration systems utilizing purified integrase lack the full fidelity of the in vivo reaction. Our results indicate that both host factors and long viral DNA substrates may be required to reconstitute an in vitro system with all the hallmarks of DNA integration in vivo.