The mouse Fv1 genetic locus controls resistance to subgroups of ecotropic, MCF, and amphotropic murine leukemia viruses (MuLVs). In addition to the four previously defined alleles of Fv1 (Fv1(n), Fv1(b), Fv1(nr), Fv1(0)), we present evidence that the novel restriction pattern characteristic of DBA/2J mice maps to the Fv1 locus and therefore represents a novel allele, here designated Fv1(d). Previous studies had demonstrated that the Fv1-mediated viral tropism is determined within the capsid protein gene, and that N- and B-tropic virus capsids differ only in two adjacent amino acids. We introduced various amino acid substitutions at these two sites in the N-tropic AKV MLV capsid gene, and typed resulting viruses for host range on cells carrying all five Fv1 alleles as well as on cells from additional wild mouse species with Fv1-like differences in virus susceptibility. Results indicate that alteration of the first of the two amino acids does not alter tropism, but alteration of the second alone is sufficient to convert the N-tropic AKV MLV to a B-tropic virus. Substitution of leucine for arginine at this site produced a virus with an unusual tropism not characteristic of any of the naturally occurring or laboratory strains of MuLV.