The 2G10 B-cell hybridoma was found to be persistently infected with reovirus serotype 3 (RV3). The persistently infected 2G10 culture produced approximately 1 x 10(8) plaque-forming units of virus per milliliter of culture lysate, and a majority of cells in the culture were infected, as determined by infectious center assay and immunocytochemistry. Cure of the persistent infection was achieved by passaging 2G10 cells for 33 days (12 passages) in medium containing polyclonal anti-RV3 antiserum and a monoclonal antibody specific for the RV3 attachment protein. After several passages in antibody-free medium, cured 2G10 cells had (1) nondetectable levels of RV3 in cell-culture lysates, (2) no infectious centers per 3 x 10(5) cells, (3) no immunocytochemically detectable RV3 antigen, and (4) no detectable reovirus-specific RNA by reverse transcription-polymerase chain reaction amplification. Additionally, mice inoculated with cured 2G10 cell lysates did not generate antibodies directed against RV3. These observations demonstrate that persistent reovirus infection of a B-cell hybridoma can be cured by passage in medium containing anti-reovirus antibodies and suggest that the maintenance of this persistent infection is dependent on horizontal cell-to-cell transmission of virus in the culture.