ABSTRACT We previously generated recombinant vesicular stomatitis viruses (VSV) based on the Indiana serotype genome which contained either the homologous glycoprotein gene from the Indiana serotype (VSIV-G ^I ) or the heterologous glycoprotein gene from the New Jersey serotype (VSIV-G ^NJ ). The virus expressing the G ^NJ gene was more pathogenic than the parental VSIV-G ^I virus in swine, a natural host (26). For the present study, we investigated the biological differences between the G ^I and G ^NJ proteins that may be related to the differences in pathogenesis between VSIV-G ^I and VSIV-G ^NJ . We show that the capacities of viruses with either the G ^NJ or G ^I glycoprotein to infect cultured cells differ depending on the pH. VSIV-G ^NJ could infect cells at acidic pHs, while the infectivity of VSIV-G ^I was severely reduced. VSIV-G ^NJ infection was also more sensitive to inhibition by ammonium chloride, indicating that the G ^NJ protein had a lower pH threshold for membrane fusion. We applied selective pressure to VSIV-G ^I by growing it at successively lower pH values and isolated variant viruses in which we identified amino acid changes that conferred low-pH-resistant infectivity. Repeated passage in cell culture at pH 6.8 resulted in the selection of a VSIV-G ^I variant (VSIV-6.8) that was similar to VSIV-G ^NJ regarding its pH- and ammonium chloride-dependent infectivity. Sequence analysis of VSIV-6.8 revealed that it had a single amino acid substitution in the amino-terminal region of the glycoprotein (F18L). This alteration was shown to be responsible for the observed phenotype by site-directed mutagenesis of a VSIV-G ^I full-length cDNA and analysis of the recovered engineered virus. A further adaptation of VSIV-6.8 to pHs 6.6 and 6.4 resulted in additional amino acid substitutions in areas of the glycoprotein that were not previously implicated in attachment or fusion.