A study of virus-specific protein synthesis in infectious pancreatic necrosis virus-infected RTG-2 cells was undertaken to find a relationship between the coding capacity of virus genome (two segments of double-stranded RNA of 2.5 X10(6) and 2.3 X 10(6) molecular weight) and the sizes and relative amounts of polypeptides in the virion and in infected cells. The time course of virus-specific protein synthesis was followed by pulse labeling infected UV-irradiated cells with [35S]methionine and analyzing the labeled proteins by polyacrylamide gel electrophoresis followed by autoradiography. Three size classes of virus-specific polypeptides were synthesized, in the same relative proportion, throughout the infectious cycle, beginning 3 h postinfection. Their designation and molecular weight was as follows: alpha1, 1000,000; alpha2, 90,000; beta1, 59,000; beta2, 56,000; gamma1, 32,000; gamma2, 30,000; and gamma3, 28,000. Experiments using amino acid analogues, protease inhibitors, ZnCl2, and supraoptimal temperatures showed that polypeptides of the beta and gamma families did not arise from the alpha polypeptides by post-translational cleavage. Slow cleavage late in the infectious cycle could be demonstrated, since during 12-h period radioactivity was chased from beta1 via beta3 to beta4 (molecular weight 50,000) and beta5 (molecular weight, 49,000). During the chase most of gamma2 was degraded, whereas radioactivity could not be chased from the remaining virus-specific polypeptides. Purified virus contained polypeptides alpha1, alpha2, beta4, beta5, and gamma1. The beta polypeptides made up over 60% of the virion proteins. The results suggest that infectious pancreatic necrosis vibrus possesses a unique mechanism for synthesis of three size-classes of proteins using mRNA transcripts from two high-molecular-weight double-stranded RNA genome segments.