Inbred mouse strains exhibit significant differences in their susceptibility to viruses in the genus Flavivirus, which includes human pathogens such as yellow fever, Dengue, and West Nile virus. A single gene, designated Flv, confers this differential susceptibility and was mapped previously to a region of mouse chromosome 5. A positional cloning strategy was used to identify 22 genes from the Flv gene interval including 10 members of the 2'-5'-oligoadenylate synthetase gene family. One 2'-5'-oligoadenylate synthetase gene, Oas1b, was identified as Flv by correlation between genotype and phenotype in nine mouse strains. Susceptible mouse strains produce a protein lacking 30% of the C-terminal sequence as compared with the resistant counterpart because of the presence of a premature stop codon. The Oas1b gene differs from all the other murine Oas genes by a unique four-amino acid deletion in the P-loop located within the conserved RNA binding domain. Expression of the resistant allele of Oas1b in susceptible embryo fibroblasts resulted in partial inhibition of the replication of a flavivirus but not of an alpha togavirus.