SUMMARY Epizootic hemorrhagic disease virus (ehdv) was isolated in Vero cell culture from the spleen and whole blood of a white-tailed deer (Odocoileus virginianus). A 10% spleen suspension caused acute hemorrhagic disease (hd) when inoculated into an experimental white-tailed deer and resulted in the recovery of ehdv from the blood of the experimental animal at 5 days after inoculation. The virus was identified as ehdv serotype 2 through indirect fluorescent antibody tests, electron microscopy, and reciprocal cross-neutralization tests. Approximately 73% (36/49) of the mule deer, 5% (2/42) of the white-tailed deer, and 79% (249/314) of the cattle samples tested from areas where hd had been reported were ehdv seropositive. Although none of the white-tailed deer was bluetongue virus seropositive, 29% of the mule deer and 3% of the cattle tested from “active” hd areas possessed bluetongue virus precipitating antibody.