We recently developed a hepatitis C virus (HCV) replication system using two cultured human cell lines: MT-BC, a human T-cell leukemia virus type I-infected cloned T cell line, and PH5CH, a non-neoplastic human hepatocyte line. In the course of the study, we found evidence that replication of hepatitis G virus (HGV), which has recently been identified, was supported in both MT-2C and PH5CH cells. When these cells were inoculated with serum 1B-3 containing both HCV and HGV obtained from a blood donor, the 5'-noncoding (5'-NC) regions of HCV RNA and HGV RNA were detected by RT-nested PCR in both cell lines more than 30 days postinoculation, and the 3'-noncoding region of HGV RNA was also detected in the both cell lines more than 30 days postinoculation. Sequence analysis of the 5'-NC region of HGV RNA revealed the quasispecies nature of HGV, although the 5'-NC region was highly conserved among HGV isolates. This HGV-infected culture system will be useful for various biological and virological studies of HGV. (C) 1997 Academic Press.