The molecular features of hepatitis C virus (HCV) replication in human fetal hepatocytes (HFHs) were addressed in this study. Using a competitive reverse-transcription polymerase chain reaction (RT-PCR) assay for the quantitation of HCV-RNA molecules, the highest level of viral replication was detected 30 days' postinfection. At this time point, viral particles of 41 to 45 nm in diameter accumulated in the cell cytoplasm. Their density in cell extracts and culture medium was distributed between heavy (1.180-1.360 g/cm3) and light fractions (1.105-1.050 g/cm3) of a sucrose gradient, while, in the serum inoculum, they had a positive fraction at 1.180 g/cm3. In infected HFHs, minus-strand HCV RNA was observed in fractions displaying a sedimentation coefficient of 28 S to 18 S, while plus-strand HCV RNA showed a peak restricted to the 21 S fraction; the HCV RNA of serum inoculum had a sedimentation coefficient of 38 to 40 S, which revealed the presence of HCV RNA of unique positive polarity. The 21 S RNA fraction of cell extracts was resistant to 20 minutes of RNase I digestion, while the same incubation time totally inactivated a comparable amount of HCV RNA purified from the serum inoculum, revealing the presence of completely and/or partially double-stranded HCV-RNA molecules in the infected cells. Detection in HFHs of replicative forms and replicative intermediates suggests that the dynamic profile of HCV replication in these cells is similar to that described in other flaviviruses.