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Two adenovirus type 5 mutants were constructed to probe the function of the virus-encoded RNA polymerase III transcripts (VA RNAs). Each mutant fails to synthesize one of the two VA RNA species. The variant that does not produce the minor VAII species grows normally. The mutant that cannot synthesize the major VAI species grows more poorly than its parent. Analysis of the mutant’s growth defect indicates that the adenovirus VAI RNA is required for efficient translation of viral mRNAs at late times after infection.