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Hepatitis C virus (HCV) is a major cause of chronic viral hepatitis. Development of anti-viral strategies has been hampered by the lack of efficient cell systems to propagate HCV in vitro. To establish a long-term culture system, we tested human hepa-toma (HuH7, HepG2) and porcine non-hepatoma (PK15, STE) cell lines, as well as several culture and infection conditions. As a marker for virus rep-lication, minus-strand HCV RNA in infected cells was detected by an enhanced detection system using nested RT–PCR followed by hybridization analysis. Short-term efficiency of HCV infection (10 days) was slightly increased by addition of polyethylene glycol (PEG) and/or dimethyl sulfoxide (DMSO) to culture media during inoculation of HuH7, PK15 and STE cells, but no augmentation in long-term culture was achieved, suggesting enhanced attachment of HCV to cells rather than more efficient infection.