Nucleic acid hybridization was used to detect epizootic hemorrhagic disease (EHD) virus serotype 1 and serotype 2 in Culicoides variipennis (Coquillett). Adult females were inoculated intrathoracically with virus, then were assayed daily for the presence of viral RNA for 2 wk, at which time maximum virus replication is likely to occur. Viral RNA of EHD serotypes 1 and 2 was first detected by hybridization on days 9 and 7 after infection, respectively, and then for up to 14 days after infection. EHD serotype 1 viral RNA was detected by hybridization in infected flies fixed in ethanol at room temperature for 7 d and in unfixed (frozen) infected flies. However, weak false positives diminished prospects for application of this method.