Viral replication and neutralization of hepatitis C viruses (HCV) have been studied using the infectious molecular clone JFH-1. By passaging JFH-1 in hepatoma cells in the absence or presence of HCV neutralizing antibodies (nAbs), we investigated the molecular mechanisms of cell-culture adaptation and sensitivity to nAbs. The cell culture-adapted JFH-1 virus (JFH-1-CA) became more sensitive to nAbs than its parental virus. Sequence analysis revealed that the predominant viruses in the JFH-1-CA population carried two mutations in their envelopes (I414T and V293A). Plasma that could neutralize JFH-1-CA was found in 2 of 7 HCV-infected individuals who have cleared the virus in blood. Plasma 226233 with a higher 50% neutralization titer was used for in vitro selection of neutralization resistant viruses. Under the increasing selection pressure of plasma 226233, the neutralizing sensitivity of JFH-1-CA decreased gradually. Two mutations (T414I and P500S) in envelope were found in all but one sequenced clones in the viral population after eight rounds of selection. Interestingly, the cell-culture adapted mutation I414T reverted back to the wild-type residue (I414) under the selection pressure. By introducing mutations at positions 414 and 500 into the JFH-1 clone, we confirmed that the T414I mutation alone can confer neutralization resistance. The results of this current study suggest that nAbs are present in a subset of HCV-infected individuals who have cleared the virus in blood. Our data also provide the first evidence that, the E2 residue P500, located within a previously identified highly conserved polyclonal epitope, may be a target for neutralizing antibodies present in individual who have spontaneously resolved the HCV infection.