Hepatitis C virus (HCV) non-structural protein 5A (NS5A) plays an essential role in viral genome replication. A series of transposon-mediated insertion mutants and deletion mutants of NS5A was used to examine the colony-forming ability of HCV subgenomic replicons encoding the mutant proteins. The results reveal that two regions of NS5A can tolerate insertions: one spanning residues 240-314, which contain the interferon sensitivity-determining region (ISDR), and the other spanning residues 349-417 at the carboxy terminus. The majority of these sites also tolerated insertion of enhanced green fluorescent protein. Furthermore, replicons encoding NS5A with deletions in ISDR or in the carboxy-terminal regions were replication-competent, indicating that these regions of NS5A are not necessary for replication. Taken together, the results suggest that the central region spanning the ISDR and the carboxy-terminal region of the molecule are dispensable for the functions of NS5A in viral genome replication.