The 5'-terminal structures of human adenovirus type 2 (Ad2) early region 2 (E2) mRNA were investigated. The E2 transcription unit has several interesting properties, including the presence of a TATA-like box that matches the consensus sequence poorly, delayed transcription during early stages of infection, and a switch in promoter recognition late after infection. E2-specific RNA, 5'-labeled in vitro to high specific activity was analyzed. Purified E2 mRNA was digested with RNase A or RNase T1 and the resulting oligonucleotides were resolved by two dimensional paper electrophoresis-homochromatography. Remarkably, as many as sixteen 5'-terminal RNase A oligonucleotides were identified and their sequences were deduced. The most common 5'-termini in the RNase A digest were p(m6)AmCp, p(m6)AmA(m)Cp, pGmA(m)Cp, and p(m6)AmG(m)Cp. Two RNase A oligonucleotides originated from the E4 promoter region, consistent with electron microscopic observations. The sequence encoding these potential initiation sites covered about 90 nucleotides. Eleven of the sequences of the 5'-terminal RNase A oligonucleotides were aligned with the Ad2 DNA sequence in the Ad2 E2 promoter region. If the heterogeneous termini in the E2 promoter region were generated by a process of transcription initiation, their existence cannot be explained by stuttering of RNA polymerase II. This suggests that the transcription of Ad early region 2 has features which differ from those of other Ad2 early gene transcription units. Perhaps this is due to the absence of a conventional TATA box which is believed to position the initiation site. Alternatively, it is conceivable that the E2 promoter represents an alternate class of RNA polymerase II promoters containing different signals with different requirements for activation and/or that an E1A gene product modifies transcription initiation.