The use of 2 strains of Friend virus, the F-S strain and its BALB/c-adapted variant, the F-B strain, permitted the identification of a second major gene distinguishing the DBA/2J (susceptible to both virus strains) and C57BL (resistant to both virus strains) mouse strains with respect to their spleen focus responses to the viruses. Progeny testing of (C57BL × DBA/2J) × DBA/2J backcross females showed that the new gene, Fv-2, segregates independently of the previously identified Fv-1 gene; Fv-2, with alleles Fv-2S (susceptible) and Fv-2r (resistant), is linked to dilute in linkage group II. Expression of the Fv-1 phenotype (relative spleen-focus response to F-S virus) is suppressed by homozygosity for the recessive Fv-2r allele, but the Fv-1 locus appears to exert little influence on the expression of the Fv-2 phenotype (spleen focus response to F-B virus). The possible relevance of these findings to the concept that spleen focus formation by Friend virus requires a helper virus is discussed.