There are two major serotypes of vesicular stomatitis virus (VSV), Indiana (VSIV) and New Jersey (VSNJV). We recovered recombinant VSIVs from engineered cDNAs that contained either (i) one copy of the VSIV G gene (VSIV-G(I)); (ii) two copies of the G gene, one from each serotype (VSIV-G(NJ)G(I)); or (iii) a single copy of the G(NJ) gene instead of the G(I) gene (VSIV-G(NJ)). The recombinant viruses expressed the appropriate glycoproteins, incorporated them into virions, and were neutralized by antibodies specific for VSIV (VSIV-G(I)), VSNJV (VSIV-G(NJ)), or both (VSIV-G(NJ)G(I)), according to the glycoprotein(s) they expressed. All recombinant viruses grew to similar titers in cell culture. In mice, VSIV-G(NJ) and VSIV-G(NJ)G(I) were attenuated. However, in swine, a natural host for VSV, the G(NJ) glycoprotein-containing viruses caused more severe lesions and replicated to higher titers than the parental virus, VSIV-G(I). These observations implicate the glycoprotein as a determinant of VSV virulence in a natural host and emphasize the differences in VSV pathogenesis between mice and swine.