Vesicular stomatitis virus (VSV) is the most common cause of vesicular disease outbreaks in livestock throughout the Western Hemisphere. Two major serotypes, Indiana and New Jersey, cause epidemic disease in pigs, cattle, and horses. We generated recombinant viruses derived from the Indiana serotype genome that were engineered to contain and express: (1) a single copy of the glycoprotein gene from the Indiana serotype (VSIV-GI); (2) a single copy of the glycoprotein gene from the New Jersey serotype (VSIV-GNJ); or (3) two copies of the glycoprotein gene, one from each of the two major VSV serotypes (VSIV-GNJGI) [Martinez I, Rodriguez LL, Jimenez C, Pauszek SJ, Wertz GW. Vesicular stomatitis virus glycoprotein is a determinant of pathogenesis in swine, a natural host. J Virol 2003;77(14):8039-47]. These recombinant viruses and a highly pathogenic New Jersey field isolate (VSNJV), from which the GNJ gene was derived, were inoculated into swine, a natural host, and the induction of neutralizing antibodies to both serotypes was analyzed. The neutralizing antibody response induced by VSIV-GI, VSIV-GNJ and VSNJV was serotype-specific, according to the glycoprotein expressed. VSIV-GNJGI expressed both glycoproteins stably through multiple rounds of replication in swine and induced neutralizing antibodies against both VSV serotypes, with a dominance of the Indiana serotype in the serological response. Pigs immunized with VSIV-GI or VSIV-GNJ were protected against homologous high dose virus challenge. Pigs inoculated with VSIV-GNJGI were protected against challenge with VSIV-GI but three of four animals developed lesions after challenge with the highly pathogenic New Jersey field isolate.