Seven isolates of the New Jersey serotype of vesicular stomatitis (VSNJ) virus were obtained from pooled specimens of phlebotomine sand flies, Lutzomyia shannoni Dyar, collected on Ossabaw Island, Chatham County, Ga., in 1989 and 1990. Three isolates, including two from males, were obtained from light-trapped sand flies in 1989. Four isolates were obtained from pools of sand flies collected from hollow trees in 1990. Three of the latter pools contained from 4.0 to 4.7 log10 of plaque-forming units of virus per ml, suggesting that the positive flies in these pools had supported VSNJ virus replication. One of these high-titered isolates was obtained from a pool of male sand flies. These data provide further support for the hypotheses that L. shannoni is a biological vector of VSNJ virus at this enzootic focus and that transovarial transmission of the virus occurs in nature.