Abstract Vesicular stomatitis New Jersey (VS NJ) virus is capable of undergoing rapid evolution in nature and therefore has the potential for antigenic variation. We selected an area of Costa Rica where VS NJ virus is enzootic to study whether this virus used the mechanism of antigenic variation to persist in nature. Three sentinel herds and three nonsentinel herds were observed from 1986 to 1988. Eleven VS N1 virus isolates were collected from naturally infected cattle. Remarkably, nine animals that were bled prior to reinfection with VS NJ virus had neutralizing antibody titers up to 1: 102,400 yet virus was isolated from, and disease was observed in, these animals. Sequence analysis of the portion of the glycoprotein gene coding for the neutralizing epitopes demonstrated that all virus isolates were 98–100% similar with no indication of specific genetic variation. The 3′ end of the glycoprotein gene also remained stable in that all isolates were again 98–100% similar in nucleotide sequence. Each isolate was neutralized to equivalent titers with monoclonal antibodies directed against four neutralizing epitopes on the glycoprotein. Additionally, preisolation sera from each animal were able to neutralize the virus that caused the subsequent disease. These results provide evidence that antigenic variation is not a mechanism used by VS NJ virus to persist in an enzootic focus of Costa Rica.