The genomic RNA of Hepatitis C virus (HCV) contains a single large open reading frame for virion Nonstructural polypeptides. HCV NS5A protein(p56) have been considered to be involved in viral RNA replication. Previous studies revealed that two phosphoproteins, p56 and p58, were produced from NS5A coding region and p58 was hyperphosphorylated form of p56. We demonstrated that kinase that is important to basal level phosphorylation was associated with C-terminus of NS5A. It is revealed that protein-protein interaction of the kinase and NS5A was not strong enough to verify the association between kinase and NS5A employing the yeast two hybrid system. Using direct in vitro kinase assay and modified affinity matrix-mediated kinase assay, we showed that there was NS5A-associated kinase activity responsible for basal phosphorylation. The deleted NS5A proteins were tested for determination of binding site. The result showed that kinase associate with and mainly phosphorylated C-terminus of NS5A. Furthermore, interaction of kinase and deleted NS5A proteins were much stronger than full size NS5A.