During vesicular stomatitis virus (VSV) infection, host protein synthesis is inhibited, while synthesis of viral proteins increases. VSV infection causes inhibition of host transcription and RNA transport. Therefore, most host mRNAs in the cytoplasm of infected cells were synthesized before infection. However, viral mRNAs are synthesized throughout infection and are newer than preexisting host mRNAs. To determine if the timing of appearance of mRNAs in the cytoplasm affected their translation during VSV infection, we transfected reporter mRNAs into cells at various times relative to the time of infection and measured their rate of translation in mock- and VSV-infected cells. We found that translation of mRNAs transfected during infection was not inhibited but that translation of mRNAs transfected prior to infection was inhibited during VSV infection. Based on these data, we conclude that the timing of viral mRNA appearance in the cytoplasm is responsible, at least in part, for the preferential translation of VSV mRNAs. A time course measuring translation efficiencies of viral and host mRNAs showed that the translation efficiencies of viral mRNAs increased between 4 and 8 h postinfection, while translation efficiencies of host mRNAs decreased. The increased translation efficiency of viral mRNAs occurred in cells infected with an M protein mutant virus that is defective in host shutoff, demonstrating that the enhanced translation of viral mRNA is genetically separable from inhibition of translation of host mRNA.