Abstract The study of adenovirus types 2 and 5 viral nuclear RNAs present at early times during a productive infection was analyzed by hybridization of pulse-labeled RNA to restriction fragments of Ad2 and Ad5 DNA. The RNA studied was the total nuclear infected cell population and the double-stranded nuclear RNAs formed after self-annealing, RNase digestion, and selection by Sephadex chromatography. In addition, the viral RNAs were synthesized in the presence and absence of cycloheximide (to restrict the infection to the early phase) at 32° and 41° (to provide control conditions for the H5ts125 mutant defective in the initiation of viral DNA synthesis at the restrictive temperature). The results of these studies established that (1) transcription of the genome is not qualitatively, but is quantitatively, affected by the use of inhibitors of viral DNA synthesis, (2) all regions of the genome are abundantly transcribed including those regions that do not code for early viral mRNA, and (3) most of these non-early messenger RNAs (which include both late mRNA and anti-sense sequences) have been post-transcriptionally modified by polyadenylation. Additionally, liquid hybridization studies of early Ad2 viral nuclear RNA to separated strands of Ad2 Eco (R1-A) fragment DNA indicate that all of these sequences (both sense and anti-sense) are represented in the RNA population and that neither the l nor r strands are preferentially transcribed. All of these results show that self-complementary RNA transcription and the transcription of non-early mRNAs is an integral part of the productive infection of human KB cells by adenovirus 2 and 5. Some of the possible functions of these sequences are discussed.