During the late stage of adenovirus 2 infection, RNA chains are initiated at a site near coordinate 16 (Evans et al., 1977) and transcribed approximately 30,000 nucleotides to the far end of the genome at coordinate 100. Late mRNAs processed from these transcripts contain a common spliced tripartite leader (Berget, Moore and Sharp, 1977; Chow et al., 1977a) encoded at approximately 16, 20 and 27, and protein coding sequences which map downstream. This report maps the late promoter and the capped 5' end of nuclear and cytoplasmic RNAs from this transciption unit, and analyzes their structures. We show that nascent RNA chains pulse-labeled in vivo are initiated at coordinate 16.5 +/- 0.5 and contain the sequences intervening between the leader segments. We map the capped 5' terminus of late nuclear transcripts at a site between 16.4 and 16.6 by aligning T1 RNAase oligonucleotides from nuclear RNA with the DNA sequence of the promoter region. The structure of the first eleven residues of the capped 5' terminus of late mRNA was determined by direct RNA sequencing. This structure corresponds exactly to a DNA sequence at coordinate 16.4 and precisely positions the mRNA cap template within the promoter region. These results suggest that the promoter and the cap template sites are coincident, and that the initiating residues of the primary transcript are precursors of the capped 5' end of mRNA. Residues removed from transcripts by splicing were identified. These plus caps were detected in large polyadenylated nuclear RNA, indicating that capping and polyadenylation can occur on unspliced molecules. Residues retained in the mRNA first leader contain a nine residue sequence adjacent to the cap which is complementary to the 3' end of 18S rRNA, suggesting that the first leader functions in ribosome binding. Nucleotide sequences from the promoter region are compared with cellular counterparts. Strong homologies at cap sites and splice points suggest that for the noted cases, the virus and cell share closely related mechanisms for mRNA 5' end synthesis and splicing.