Mouse plasmacytoma ascites tumor cells (MOPC 460) were efficiently infected with encephalomyocarditis virus. Inhibition of host protein synthesis was evident after 2 h and complete by 4 h postinfection. The mechanism by which virus infection results in inhibition of host cell protein synthesis was studied in vitro. Cell-free protein-synthesizing systems, prepared from uninfected and infected cells, were found to be equally active with respect to their abilities to translate cellular and viral mRNAs. The plasmacytoma cell-free system was also shown to be insensitive to the addition of double-stranded viral RNA. Host cellular mRNA was isolated from uninfected and infected cells. No difference in the amount or size distribution of the mRNA was detected. However, the mRNA from infected cells was translated only 46 to 49% as actively as that from uninfected cells. mRNA isolated from cells in which initiation of protein synthesis was inhibited with pactamycin was similarly inactivated. Simultaneous addition of viral RNA and cellular mRNA to the plasmacytoma cell-free system resulted in a complete suppression of the translation of the cellular message, whereas viral RNA was translated normally.