In adenovirus type 5-infected cells, RNA polymerase Ⅲ transcription of a gene superimposed on the 5 end of the E2E RNA polymerase Ⅱ transcription unit produces two small (<100-nucleotide) RNAs that accumulate to low steady-state concentrations (W. Huang, R. Pruzan, and S. J. Flint, Proc. Natl. Acad. Sci. USA 91: 1265-1269, 1984). To gain a better understanding of the function of this RNA polymerase III transcription, we have examined the properties of the small E2E RNAs and E2E RNA polymerase III transcription in more detail. The accumulation of cytoplasmic E2E RNAs and the rates of E2E transcription by the two RNA polymerases during the infectious cycle were analyzed by using RNase T1 protection and run-on transcription assays, respectively. Although the RNA polymerase III transcripts were present at significantly lower concentrations than E2E mRNA throughout the period examined, E2E transcription by RNA polymerase Ⅲ was found to be at least as efficient as that by RNA polymerase Ⅱ. The short half-lifes of the small E2E RNAs estimated by using the actinomycin D chase method appear to account for their limited accumulation. The transcription of E2E sequences by RNA polymerase Ⅱ and Ⅲ in cells infected by recombinant adenoviruses carrying ectopic E2E-CAT (chloramphenicol transferase) reporter genes with mutations in E2E promoter sequences was also examined. The results of these experiments indicate that recognition of the E2E promoter by the RNA polymerase Ⅱ transcriptional machinery in infected cells limits transcription by RNA polymerase Ⅲ, and vice versa. Such transcriptional competition and the properties of E2E RNAs made by RNA polymerase Ⅲ suggest that the function of this viral RNA polymerase Ⅲ transcription unit is unusual.