Closed circular viral DNA of simian sarcoma virus (SSV) and simian sarcoma-associated virus (SSAV) obtained from acutely infected dog cells was purified on preparative agarose gels, cleaved with EcoRI, and cloned in the phage lambda vector Charon 21A. The cloned 9-kilobase SSAV genome (B11) has the same restriction map as the bulk of the unintegrated linear SSAV DNA intermediate. Heteroduplex analysis between an SSV clone (lambda-C60) and an SSAV clone (lambda-B11) showed two substitution loops and one deletion loop. By using detailed restriction enzyme mapping and electron microscopic analysis, we showed that one of the substitution loops corresponds to an inversion of one of the two long terminal repeat units and adjacent cellular sequences in C60. The other substitution loop mapped close to the 3' long terminal repeat. At least part of this region was shown to contain SSV-specific sequences not shared by SSAV. The 1.9-kilobase deletion mapped at 3.5-5.5 kilobases of the linear SSAV genome, corresponding to most, if not all, of the pol gene.