Replication of the plus-stranded RNA genome of hepatitis C virus (HCV) occurs in a membrane-bound replication complex consisting of viral and cellular proteins and viral RNA. NS5B, the RNA polymerase of HCV, is anchored to the membranes via a C-terminal 20-amino-acid-long hydrophobic domain, which is flanked on each side by a highly conserved positively charged arginine. Using a genotype 1b subgenomic replicon (V. Lohmann, F. Korner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartensclager, Science 285:110-113, 1999), we determined the effect of mutations of some highly conserved residues in this domain. The replacement of arginine 570 with alanine completely abolished the colony-forming ability by the replicon, while a R591A change was found to be highly detrimental to replication, viability, and membrane binding by the mutant NS5B protein. Mutations of two other highly conserved amino acids (L588A and P589A) reduced but did not eliminate colony formation. It was of interest, if specific amino acid residues play a role in membrane anchoring of NS5B and replication, to determine whether a complete exchange of the NS5B hydrophobic domain with a domain totally unrelated to NS5B would ablate replication. We selected the 22-amino-acid-long hydrophobic domain of poliovirus polypeptide 3A that is known to adopt a transmembrane configuration, thereby anchoring 3A to membranes. Surprisingly, either partial or full replacement of the NS5B hydrophobic domain with the anchor sequences of poliovirus polypeptide 3A resulted in the replication of replicons whose colony-forming abilities were reduced compared to that of the wild-type replicon. Upon continued passage of the replicon in Huh-7 cells in the presence of neomycin, the replication efficiency of the replicon increased. However, the sequence of the poliovirus polypeptide 3A hydrophobic domain, in the context of the subgenomic HCV replicon, was stably maintained throughout 40 passages. Our results suggest that anchoring NS5B to membranes is necessary but that the amino acid sequence of the anchor per se does not require HCV origin. This suggests that specific interactions between the NS5B hydrophobic domain and other membrane-bound factors may not play a decisive role in HCV replication.