Protein synthesis in the murine hepatitis virus JHM-infected cells was temporarily inhibited by hypertonic shock. When the cells were returned to isotonic medium the synthesis of six virus-specific polypeptides, 150K, 65K, 60K, 30K, 23K and 14K was reinitiated simultaneously. Polyadenylated RNA isolated from the cytoplasm or polysomes of infected cells was translated in vitro and the products included polypeptides with molecular weights (mol. wt.) of 120,000, 60,000, 30,000, 23,000 and 14,000. Immunoprecipitation and fingerprinting of [35S]methionine-containing tryptic peptides showed that the 60,000 and 23,000 mol. wt. products were identical to the 60K and 23K polypeptides found in infected cells; the 120,000 mol. wt. product showed identity with the 150K intracellular polypeptide and a virus-specific 120K polypeptide synthesized in tunicamycin-treated cells. Two-dimensional polyacrylamide gel electrophoresis strongly suggested that the 30,000 and 14,000 mol. wt. products are equivalent to virus-specific 30K and 14K intracellular polypeptides. [3H]Uridine-labelled polyadenylated virus RNA was isolated from infected cells and sedimented in sucrose gradients containing formamide. The distribution in the gradient of each of the previously identified virus RNAs was determined by gel electrophoresis and gradient fractions enriched for each RNA were translated in vitro. The 120,000, 60,000, 30,000, 23,000 and 14,000 mol. wt. polypeptides were found to be encoded by mRNAs 3, 7, 2, 6, and 4 or 5 respectively. These results demonstrate that the virus-specific polypeptides in JHM-infected cells are encoded in separate subgenomic mRNAs and are translated independently. The assignment of coding functions and the known sequence relationships of JHM RNAs permitted a gene order to be deduced.