Induction of erythroleukemia in mice by the replication-defective spleen focus-forming virus (SFFV) relies on the presence of a helper virus to deliver the SFFV genome to erythroid target cells. Pseudotyping studies with different ecotropic murine leukemia viruses (MuLV) have shown that SFFV pseudotyped with Akv, the endogenous ecotropic virus of AKR mice, inefficiently gives rise to virus-induced erythroid bursts (vBFU-E) in vitro and fails to cause erythroleukemia in mice when compared to SFFV pseudotyped with Friend or Moloney MuLV. In order to locate the region(s) of the Akv genome responsible for its inability to act as a helper for SFFV, six different Moloney MuLV chimeras containing Akv envelope sequence substitutions were constructed. Virions with the chimeric envelopes were used to pseudotype SFFV and the complexes were analyzed for their ability to induce vBFU-E in vitro and erythroleukemia in mice. SFFV preparations pseudotyped with three of the constructs containing chimeric envelope genes efficiently gave rise to vBFU-E as did SFFV pseudotyped with Moloney MuLV. SFFV pseudotypes generated from the other three constructs, which all share a common 304-bp region located near the center of the Akv gp70 coding region, and Akv gave rise to very few vBFU-E. However, all SFFV preparations, with the exception of SFFV pseudotyped with Akv, induced erythroleukemia in mice. The results suggest that specific sequences present in the envelope gene of Akv are responsible for the inefficiency of the virus to infect erythroid target cells for SFFV, but additional Akv sequences outside those used in this study affect the ability of the Akv/SFFV virus complex to cause erythroleukemia in mice.