A complete cDNA clone of the larger A segment of the genome of infectious pancreatic necrosis virus (IPNV) was expressed in Escherichia coli in an effort to develop a vaccine for IPNV in fish. When the cDNA insert was positioned in the correct orientation to the pUC19 lacZ promoter, the viral proteins VP2, NS, and VP3 were synthesized and processed as observed in infected cells. When the insert was placed in the opposite orientation, VP3 and a 38-kDa virus-specific polypeptide were also synthesized. In addition, specific deletions made from the 3' end into the NS gene of the cloned A segment led to inactivation of the NS proteolytic activity and subsequently, the synthesis of an unprocessed VP2-NS polyprotein precursor. Antiserum to this polyprotein distinguished NS (28.5 kDa) from VP3 (31 kDa) and led to the identification of a previously undescribed 38-kDa virus-specific polypeptide in infected cells. Thus, both internal translational initiation and proteolytic cleavage could lead to the synthesis of VP2, NS, and VP3 from a single mRNA with a single open reading frame. A trpE expression vector, pATH2, was used to synthesize large quantities of the A-segment-encoded proteins in bacteria. The resulting bacterial lysate was very effective in inducing protective immunity in rainbow trout fry.