A panel of neutralizing and nonneutralizing monoclonal antibodies (MAbs) to the Indiana strain of vesicular stomatitis virus (VSV-IND) were used to select nonbinding VSV-IND mutants. In addition, virus was passaged against high titered polyclonal antisera to select for poorly neutralized virus mutants. Nucleic acid sequencing localized mutations in the surface spike glycoprotein (G protein) sequence which were associated with decreased neutralization by polyclonal antisera and with nonbinding by neutralizing and nonneutralizing MAbs. The major neutralization epitope, the A epitope, is composed of at least two regions that are widely separated in the primary G protein sequence. We found evidence for both continuous and discontinuous determinants within the A epitope and found one strongly selected region that may function as an antigenic loop. The high degree of sequence homology between VSV-IND and the other major VSV serotype, VSV-New Jersey (VSV-NJ) allowed us to predict some of the neutralization epitopes of VSV-New Jersey. Alignment of VSV and rabies virus sequences revealed that major neutralizing epitopes in VSV correspond to sites of carbohydrate attachment in rabies. This may be of significance in the evolution of rhabdoviruses.