We have developed a cell-free system for studying the integration of retroviral DNA. In our assay, amber mutations in a bacteriophage lambda genome that serves as the target for integration are suppressed by integration of an MLV derivative that carries the E. coli supF gene. The structure of the reaction products is that expected from an authentic MLV integration reaction. Linear viral DNA from the cytoplasm of infected cells serves as a precursor, though not necessarily the immediate precursor, to the provirus integrated in vitro. The viral DNA in the infected cell appears to be tightly associated with the enzymatic machinery required for its integration. Supercoiling, chromatin structure, transcription, and replication are not required of the target DNA. Since no high-energy cofactor is necessary, the DNA breakage and joining steps in the integration reaction are probably coupled.