The sensitivity of different assay systems for detecting low concentrations of bluetongue virus (BTV) were compared. These assays included blind passage on baby hamster kidney (BHK-21) cells and on cattle pulmonary artery endothelial (CPAE) cells, immunoperoxidase staining of cells on multiwell slides, and cDNA/RNA hybridization of BTV infected cells. Nine serial 10-fold dilutions of a cell culture-adapted BTV serotype 11 were tested (each dilution was treated as a separate sample) in all assays. Visual inspection for cytopathic effects (CPE) during 3 passages in BHK-21 cells detected samples that contained greater than or equal to 3 plaque forming units (PFU)/ml of BTV. Evidence of CPE during 3 passages in CPAE cells detected samples that contained greater than or equal to 0.3 PFU/ml of BTV. A limit of detection (greater than or equal to 0.3 PFU/ml) was obtained faster by immunoperoxidase staining of BTV-inoculated CPAE cells on multiwell slides and incubated for 3 days. The cDNA/RNA hybridizations of CPAE and BHK-21 cells incubated for 2 or 3 days, respectively, with BTV dilution samples detected samples that contained greater than or equal to 30 PFU/ml. Of the assay systems examined, immunoperoxidase staining of CPAE cells on multiwell slides inoculated with cell culture-adapted BTV was the most sensitive and fastest assay for definitive virus identification.