Fv-4 is a mouse gene that dominantly confers resistance to infection by ecotropic murine leukemia virus (MuLV). The Fv-4′ env antigen that binds to the cell surface of Fv-4′-bearing C3H cells was found in sera from normal Fv-4′ -bearing C4W mice. The serum Fv-4′ env antigen binds to ecotropic MuLV receptors, shown by specific binding to transfectant cells expressing ecotropic MuLV receptors but not to parental mink cells. To determine whether the binding of Fv-4′ env antigen to the putative MuLV receptors would block FLV infection, C3H thymocytes or spleen cells that had been preincubated with C4W serum were mixed with FLV and the subsequent production of MuLV specific antigens was examined. C3H thymocytes or spleen cells treated with C4W serum became refractory to binding by FLV. These results provide evidence that the Fv-4′ env antigen is released from C4W-derived cells in vivo and binds to cells expressing surface receptors for ecotropic MuLV, thereby protecting them from infection with FLV. The implication of these findings for gene therapy of retrovirus-induced disease such as acquired immune deficiency syndrome (AIDS) is discussed.