The pathogenic Friend virus complex is of considerable interest in that, although members of this group are genetically related, they differ markedly in biochemical and biological properties. Heteroduplex mapping of molecular clones of the Friend virus complex, which includes the replication-competent ecotropic Friend murine leukemia virus (F-MuLV) and mink cell focus-forming virus (F-MCF) and replication-defective polycythemiaand anemia-inducing strains of spleen focus-forming virus (SFFVp and SFFVa, respectively), was employed to provide insight into the molecular basis of their relationships. In heteroduplexes of F-MuLV x F-MCF, a major substitution of 0.89 kilobases in the env gene of F-MCF was discerned. Heteroduplexes of SFFVp x F-MuLV or F-MCF and SFFVa x F-MuLV or F-MCF showed several major deletions in the pol gene region and a single major deletion in the 3' half of the env gene region of SFFVp and SFFVa. A major substitution of 0.89 kilobases was mapped to the 5' end of the env deletion of SFFVp and SFFVa in heteroduplexes with F-MuLV, similar to that seen in F-MuLV x F-MCF heteroduplexes. In contrast, this env gene region was totally homologous in F-MCF x SFFVp or SFFVa and SFFVp x SFFV. heteroduplexes. Our results suggest that (i) both SFFVp and SFFVa lack part of the env gene at its 3' end, corresponding to the p15(E) coding region, (ii) major deletions occur in the pol and env genes which account for the replication defectiveness of SFFVp and SFFVa, (iii) minor substitutions occur in the gag gene region of SFFVa that are not present in SFFVp, F-MuLV, or F-MCF, (iv) a major substitution exists in the gp7O region of the env gene between F-MuLV and F-MCF that probably accounts for the differences in their host range specificities, (v) this substitution in F-MCF is identical to the gp7O part of the gp52 coding region of SFFVp and SFFVa, and (vi) heteroduplexes to F-MCF show unambiguously that no additional large substitutions are present in SFFVp or SFFVa that could account for differences in their leukemogenicity. The original isolate of Friend virus described in 1957 caused acute erythroblastosis associated with anemia (13). Passage of this Friend virus stock in several laboratories has resulted in the identification of several pathogenic strains that differ in their biological and biochemical properties (for a review, see reference 44). For example, Axelrad and Steeves (2) and Mirand et al. (24) described strains of Friend virus that caused polycythemia in susceptible mice, rather than anemia as originally described (13). Although all of these "strains" produce rapid erythroid proliferation, erythroleukemia, splenomegaly, and splenic foci, they can be distinguished by diagnosis of the terminal stages of their disease as being accompanied by polycythemia or anemia (2, 13, 24, 37, 44). The polycythemia and anemia strains of the Friend virus complex are each composed of at least two distinct viral components: (i) a replication-competent, ecotropic helper type C virus (F-MuLV) (23, 26, 38, 43, 45, 46) a...