The non-structural protein NS4B of the Hepatitis C virus (HCV) is an integral membrane protein located in the endoplasmic reticulum (ER). Although the function of the NS4B in the viral life cycle is unknown a critical role in replication has been indicated. In order to investigate which components are involved we initially introduced restriction sites near the extremities of the NS4B in a subgenomic replicon that resulted in the alterations of six amino acid residues. This totally abolished replication. We subsequently introduced 14 single point mutations into different regions of NS4B based on the current topology model. One mutation abolished replication, while most conferred reduced replicon establishment and one mutation resulted in improved efficiency. Neither the protein processing nor the membrane altering capacity of NS4B was affected. Surprisingly, mutations situated in the ER lumen also conferred strong effects, despite the fact that replication occurs on the cytosolic side of the ER membrane. We conclude that the molecular integrity of NS4B is vital for HCV replication. Our results suggest that NS4B interacts with itself and with other viral and cellular factors, and may carry intrinsic capacities in order to allow replication.