The integration sites for viral DNA in cells infected with Moloney murine leukemia virus (M-MuLV) were studied by restriction endonuclease cleavage of cellular DNA followed by electrophoresis in agarose gels, blot transfer to nitrocellulose, and detection by M-MuLV-related sequences by hybridization with high-specific-activity 32P-labeled M-MuLV complementary DNA. When EcoRI was used to cleave cellular DNA, numerous DNA fragments with sequence homology to M-MuLV were detected in uninfected mouse cell DNA. These endogenous sequences are mouse specific since they are not detectable in rat cell DNA, and are related to the 38S genomic RNA of M-MuLV. Infected cells contain additional M-MuLV-specific DNA fragments which are not detected in uninfected cells. Different patterns of M-MuLV-specific DNA fragments were detected in each cloned infected line examined. These data suggest the existence of multiple sites for integration of M-MuLV DNA in infected mouse fibroblasts. Cleavage of infected cell DNA with BamHI, which cleaves M-MuLV viral DNA at least twice, released the internal BamHI B fragment from each infected line, confirming the presence of integrated M-MuLV DNA sequences in each infected cell line which retain some features of the sequence organization of unintegrated M-MuLV DNA.