The longest DNA molecules synthesized by endogenous reverse transcription in detergent-permeabilized Moloney murine sarcoma virus (Mo-MSV) virions (clone G8-124) are double-stranded DNA molecules of 5,8 kilobase pairs (kbp). This DNA species has been purified by sedimentation of total in vitro synthesized Mo-MSV DNA through neutral sucrose gradients. A physical map of the positions of the cleavage sites for a series of restriction endonucleases has been derived for this 5.8 kbp DNA. Mo-MSV DNA synthesized in vitro was found to induce morphological transformation of NIH-3T3 mouse fibroblasts upon transfection. The foci had a morphology indistinguishable from that of Mo-MSV-induced foci, and the induced transformed phenotype was stable. The 5.8 kbp double-stranded DNA (dsDNA) purified by agarose gel electrophoresis also induced focal transformation. Furthermore, gel-purified, restriction endonuclease-generated fragments of 5.8 kbp dsDNA containing the region from 2.8--4.9 kbp on the physical map of Mo-MSV DNA were able to induce foci. In contrast, endonuclease-generated DNA fragments lacking this region on the map were unable to transform cells upon transfection. When transformants derived by transfection with 5.8 kbp dsDNA were infected with Moloney murine leukemia virus (Mo-MLV) helper virus, Mo-MSV was rescued from a small portion of these cells, suggesting the establishment of the complete viral genome in these cells. One Mo-MSV DNA fragment, spanning 2.8--4.9 kbp on the physical map, was generated by cleavage of 5.8 kbp DNA with endonucleases Hind III + Sal I and currently represents our maximum estimate for the size of the transforming region of the Mo-MSV genome. This fragment includes the Mo-MSV sequences which are found in the DNA of uninfected mouse cells.