The time course of appearance of transcriptional activity from five early adenovirus type 2 transcription units has been determined. RNA complementary to region 1A (1-4.4 map units), the first region to be transcribed, was detectable at 45 min after infection; a maximal rate of RNA synthesis was reached at 3 h after infection and was maintained thereafter for at least 6 h. RNA from region 2 (75-56 map units), which encodes the mRNA for the 72,000-dalton DNA-binding protein, was the last to be synthesized; transcription commenced at about 2 h postinfection, reached a maximum at 7 h, and then declined. Transcription of regions 3 (76-86 map units) and 4 (99-91 map units) reached a maximal value at 3 h postinfection. The rates of RNA synthesis from these regions then declined over the next 6 h. The decline of transcription from regions 2 and 4 appeared to be a specific repression of these transcription units. The repression did not occur in the absence of protein synthesis, suggesting that a viral protein might be involved. Transcription of all early regions was initiated and continued for at least 2 to 3 h in cells that were treated with cycloheximide or emetine before and during infection, suggesting that at least the initiation of RNA synthesis from the five early adenovirus type 2 transcription units does not depend on the formation of a viral protein. Moreover, mRNA was formed in the absence of protein synthesis that hybridized to DNA fragments representing each of the five early transcription units. The increase in mRNA accumulation in the presence of cycloheximide (or emetine) does not appear to be due to increased RNA synthesis; thus, either increased mRNA stability or increased efficiency of nuclear RNA processing must occur.