A comparison was made of bacteriophage MS2 RNA translation in infected Escherichia coli cells and in a defined cell-free system. A number of temperature-sensitive mutants were used as hosts for viral RNA translation at permissive and restrictive temperatures. The amount of viral coat protein synthesis was determined after gel electrophoresis of proteins from the cell lysates. These results were compared to those obtained with cell-free translation assays conducted with ribosomes isolated from the same mutants. Compared with control cells, a reduced activity in vivo and in vitro was found for each mutant examined at elevated temperatures. A good correlation between the two types of translational assays was observed. These findings are discussed in terms of the translational defects known to be a characteristic of some of these mutant strains.