The adenovirus VAI RNA is a small polymerase III-transcribed species that is required for optimal translation of mRNAs late after infection. Mutant dl331 fails to produce this RNA species and, as a result, grows poorly. Mutant-infected cells contain normal levels of late mRNAs, but reduced levels of polypeptides are synthesized late after infection. Translational elongation occurs at normal rates in mutant, as compared to wild-type, virus-infected cells. Initiation of translation occurs with reduced efficiency in dl331 -infected cells. VAI RNA is required for formation of a stable 48S preinitiation complex and very likely functions to facilitate the interaction between 43S preinitiation complex and mRNA to form the 48S species.