AIM: To develop a cell culture system capable of producing high titer hepatitis C virus (HCV) stocks with recombinant vaccinia viruses as helpers.METHODS: Two plasmids were used for the generation of recombinant HCV: one containing the full-length HCV cDNA cloned between T7 promoter and T7 terminator of pOCUS-T7vector, and the other containing the HCV polyprotein open reading frame (ORF) directly linked to a vaccinia late promoter in PSC59. These two plasmids were co-transfectedinto BHK21 cells, which were then infected with vTF7-3recombinant vaccinia helper viruses.RESULTS: After 5 d of incubation, approximately 3.6×107copies of HCV RNA were present per milliliter of cell culture supatant, as detected by fluorescence quantitative RT-PCR (FQ-PCR). The yield of recombinant HCV using this cellsystem increased 100- to 1 000- fold compared to in vitrotranscribed HCV genomic RNA or selective subgenomic HCV RNA molecule method.CONCLUSION: This cell culture system is capable of producing high titer recombinant HCV.