Full-length and truncated genome segment A-specific infectious pancreatic necrosis virus cDNA was subcloned into plasmid transcription vectors, and runoff transcripts were produced in vitro. These transcripts were translated in cell-free rabbit reticulocyte lysates and the translation products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Virus-specific polypeptides were gel purified and mapped by partial proteolysis with N-chlorosuccinimide and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide profiles were compared with those of the corresponding polypeptides purified from infectious pancreatic necrosis virus-infected cells or prepared by in vitro translation of denatured genomic RNA. The cDNA directed the synthesis of authentic pVP2, VP3, and NS polypeptides as well as a number of previously undescribed polypeptides. A 101,000-molecular-weight polypeptide was isolated and shown to be the unprocessed infectious pancreatic necrosis virus polyprotein. The NS polypeptide appears to be a virus-encoded protease responsible for the cleavage of pVP2 from the polyprotein. The carboxy terminus of NS was mapped to within three or four amino acids on the polyprotein. The most likely internal translation start sites responsible for NS and VP3 production in vitro were also mapped.