Recombinant vaccinia viruses were used to study the processing of hepatitis C virus (HCV) nonstructural polyprotein precursor. HCV-specific proteins and cleavage products were identified by size and by immunoprecipitation with region-specific antisera. A polyprotein beginning with 20 amino acids derived from the carboxy terminus of NS2 and ending with the NS5B stop codon (amino acids 1007 to 3011) was cleaved at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B sites, whereas a polyprotein in which the putative active site serine residue was replaced by an alanine remained unprocessed, demonstrating that the NS3-encoded serine-type proteinase is essential for cleavage at these sites. Processing of the NS3'-5B polyprotein was complex and occurred rapidly. Discrete polypeptide species corresponding to various processing intermediates were detected. With the exception of NS4AB-5A/NS5A, no clear precursor-product relationships were detected. Using double infection of cells with vaccinia virus recombinants expressing either a proteolytically inactive NS3'-5B polyprotein or an active NS3 proteinase, we found that cleavage at the NS4A/4B, NS4B/5A, and NS5A/5B sites could be mediated in trans. Absence of trans cleavage at the NS3/4A junction together with the finding that processing at this site was insensitive to dilution of the enzyme suggested that cleavage at this site is an intramolecular reaction. The trans-cleavage assay was also used to show that (i) the first 211 amino acids of NS3 were sufficient for processing at all trans sites and (ii) small deletions from the amino terminus of NS3 selectively affected cleavage at the NS4B/5A site, whereas more extensive deletions also decreased processing efficiencies at the other sites. Using a series of amino-terminally truncated substrate polyproteins in the trans-cleavage assay, we found that NS4A is essential for cleavage at the NS4B/5A site and that processing at this site could be restored by NS4A provided in cis (i.e., together with the substrate) or in trans (i.e., together with the proteinase). These results suggest that in addition to the NS3 proteinase, NS4A sequences play an important role in HCV polyprotein processing.