DBA/2 (D2) mice are susceptible and C57BL/6 (B6) mice are resistant to lethal mousepox. A congenic resistant strain, D2.B6-Rmp-4r (D2.R4), was developed by serially backcrossing male mice that survived ectromelia virus infection with D2 mice, beginning with (B6 x D2)F1 mice. Male D2.R4 mice were at least 300-fold more resistant to lethal mousepox than male D2 mice. Female D2.R4 mice were 100-fold more resistant than male D2.R4 mice and 500-fold more resistant than female D2 mice. Neonatal gonadectomy prevented development of resistance in D2.R4 mice of both sexes. Differences in resistance between strains and between sexes correlated with restriction of virus replication in spleen and liver, but gender differences were less evident in liver than in spleen. High-resolution interval mapping of the 19 autosomes of D2.R4 mice using dispersed informative microsatellites as marker loci revealed a segment of distal chromosome 1 to be of B6 origin. Haplotypes for a marker locus, D1Mit57, from the differential segment were determined in (D2.R4 x D2)F1 x D2 backcross mice, which were then infected with ectromelia virus. Significantly more heterozygotes than homozygotes survived ectromelia virus infection in both sexes. Whereas nearly all surviving males were heterozygotes, 44% of surviving females were homozygotes. These results indicate that resistance in D2.R4 mice is determined by a gonad-dependent gene on distal chromosome 1, provisionally named Rmp-4, and by an ovary-dependent factor that is not genetically linked to Rmp-4.