The NS5A protein of hepatitis C virus is believed to be an integral part of the viral replicase. Despite extensive investigation, the role of this protein remains elusive. Only limited biochemical characterization of NS5A has been performed, with most research to date involving the myriad of host proteins and signaling cascades that interact with NS5A. The need for better characterization of NS5A is paramount for elucidating the role of this protein in the virus life cycle. Examination of NS5A using bioinformatics tools suggested the protein consisted of three domains and contained an unconventional zinc binding motif within the N-terminal domain. We have developed a method to produce NS5A and performed limited proteolysis to confirm the domain organization model. The zinc content of purified NS5A and the N-terminal domain of NS5A was determined, and each of these proteins was found to coordinate one zinc atom per protein. The predicted zinc binding motif consists of four cysteine residues, conserved among the Hepacivirus and Pestivirus genera, fitting the formula of CX17CXCX20C. Mutation of any of the four cysteine components of this motif reduced NS5A zinc coordination and led to a lethal phenotype for HCV RNA replication, whereas mutation of other potential metal coordination residues in the N-terminal domain of NS5A, but outside the zinc binding motif, had little effect on zinc binding and, aside from one exception, were tolerated for replication. Collectively, these results indicate that NS5A is a zinc metalloprotein and that zinc coordination is likely required for NS5A function in the hepatitis C replicase.