The structure of r-strand-specific RNAs encoded between coordinates 26 and 32 on the adenovirus type 2 genome was mapped by a combination of S1 endonuclease analysis, primer extension, and in vitro transcription. The region includes the third leader segment (coordinates 26.8 to 27.0), the genes for the low-molecular-weight virus-associated RNAs (VA RNAs) (coordinates 29.5 to 30.7), and the amino-terminal end of the gene for the L1 52,000-55,000 polypeptide (coordinates 30.7 to 32.1). The positions at which the tripartite leader was attached to the three longest L1 mRNAs were mapped at the nucleotide level. The leader splice junction of species L1a was located at coordinate 26.8 and coincided with the 3' splice site for the third leader segment, whereas the leader-body splice junction of species L1b and L1c were located at coordinates 29.0 and 30.7, respectively. No protein products have so far been assigned to the L1a and L1b mRNAs, although it can be predicted from the nucleotide sequence that species L1b encodes a 8,300 polypeptide. The third RNA, species L1c, encodes the well-characterized 52,000-55,000 polypeptide. It was also shown that a previously unidentified class of VA RNAs exists predominantly in the poly(A)-fraction of late RNA preparations. These RNAs are heterogeneous in length (up to 3,000 nucleotides) because of irregular transcription termination and have 5' ends which map precisely to the initiation sites for VA RNAI and VA RNAII transcription. Finally it was shown that an RNA with a 5' end coinciding with the 5' splice site for the third leader segment exists in the poly(A)-fraction of late cytoplasmic RNA. This RNA species might represent an excised intron.