The serogroup specificity of the bluetongue virus (BTV) NS1 and VP3 gene probes was confirmed by means of northern blot hybridization. Under high-stringency conditions both probes hybridized to 22 BTV serotypes (18 South African serotypes, BTV3 from Cyprus and BTV16 from Pakistan) but not to serotypes that originate from Australia and India. Furthermore, NS1 gene probes of BTV and African horsesickness virus (AHSV) were used in a dot-spot in situ hybridization procedure to differentiate between BTV and AHSV in co-infected cell cultures. The method detects viral RNA directly i glutaraldehyde-fixed infected cell cultures without prior nucleic-acid extraction or purification. AHSV could be detected in cells infected with AHSV at a multiplicity of infection of 10(-4) PFU/cell in the presence of a hundred excess of co-infecting BTV. The method may have an application in epidemiological surveys to detect different orbiviruses in the same Culicoides population.