We have employed a pET-ubiquitin expression system to produce two his-tagged forms of hepatitis C virus (HCV) non-structural protein 5A (NS5A) in Escherichia coli. One derivative contains the full-length protein extended to include a carboxy-terminal hexahistidine tag; the other derivative contains an amino-terminal hexahistidine tag in place of the 32 amino acid amphipathic helix that mediates membrane association. At least 1 mg of each derivative at a purity of 90% could be produced from a 1-L culture. The purified derivatives produced high titer antibody that recognized both p56 and p58 forms of NS5A in Huh-7.5 cells expressing an HCV subgenomic replicon. The NS5A derivatives were efficiently phosphorylated by casein kinase II, leading to at least 5 mol of phosphate incorporated per mole of protein. Interestingly, this level of phosphorylation did not alter the migration of the protein in an SDS-polyacrylamide gel, suggesting that hyperphosphorylation alone is not sufficient to generate the p58 form of NS5A observed in Huh-7 cells. Neither NS5A derivative was capable of inhibiting the eIF2alpha-phosphorylation activity of the activated form of the double-stranded RNA-activated protein kinase, PKR, suggesting that NS5A phosphorylation may be required for this function of NS5A. However, both unphosphorylated derivatives were shown to interact with NS5B, the HCV RNA-dependent RNA polymerase, in solution by using a novel kinase-protection assay. The availability of purified HCV NS5A will permit rigorous biochemical and biophysical characterization of this protein, ultimately providing insight into the function of this protein during HCV genome replication.