By constructing deletion mutations in cloned adenovirus types 2 and 5 VAI genes and measuring the ability of altered templates to direct transcription of VAI RNA in HeLa cell extracts, we have located two transcriptional control regions. The first is an intragenic region located between positions +9 and +72 relative to the 5' end of the VAI(A) RNA. Those deletions examined within these sequences abolished the transcription of mutant templates in HeLa cell extracts. The second control region includes 5' flanking sequences which abut the VAI coding region. Mutations here can reduce the efficiency with which the VAI gene is transcribed. Nucleotide sequence similarities were noted on comparison of the VAI intragenic control region to tRNA sequences, which lead us to speculate that the transcriptional regulation of these two types of genes may be quite similar; the adenovirus VA genes may even have evolved from a tRNA gene(s).