Multiplex reverse transcription-polymerase chain reaction for the simultaneous detection of HCV and HGV RNA has been established, in which primers were deduced from high conservative region of HCV and HGV and PCR amplicons were detected by microtiter plate reverse hybridization with HCV or HGV specific probe. Sequence analyses showed that amplicons of HCV were 93.1%-94.1% and 92.5%-93.7% of nucleotide homology compared with Takamizawa and Choo, and amplicons of HGV were 90.7%-92.5%, 92.0%-92.1% and 94.3%-94.5% of nucleotide homology with Simons, Linnen and Chang. The detective sensitivity was 100 times over that of electrophoretic assay with single amplification. CVs of HCV and HGV were 8.9% and 9.8%, respectively, and the optimal concentration of NaOH for hybridization was 0.1-0.15 mol/L. The optimal time for hybridization was 30-50 minutes. The results of detection with multiplex PCR showed the presence of infection with HCV or HGV alone and coinfection with both % them.