The reverse transcription followed by the polymerase chain reaction (RT-PCR) technique was applied to the detection of African horsesickness virus (AHSV) using primers specific for attenuated AHSV serotype 4 segment 5 (NS1 gene). Total RNA which contains both messenger RNA and genomic dsRNA was extracted by the acid guanidinium-phenol-chloroform method from the AHSV infected Vero cells and was used as templates to optimize the RT-PCR. A pair of primer (NP2-NP32) amplified the product of the expected size from all serotypes of attenuated AHSV when four pairs of primers were tested. Using this primer pair, no RT-PCR product was detected from the RNA samples extracted from ten other orbiviruses infected cells and their virions. In addition, RT-PCR using a serial dilution of RNA samples suggested that AHSV was efficiently detected from 1 to 2 cells of the cell monolayer infected with 10(6) TCID50 of AHSV. The RT-PCR concerning with total RNAs of AHSV NS1 gene was found to be a specific and sensitive method for the detection of AHSV.