Adenovirus 40 (Ad40) is defective for growth in tissue culture but is complemented when the Ad2/5 or Ad12 E1B 55K protein is supplied in trans. Ad40 E1B mRNA has not been detected in E1-transformed cells, or at early times in lytically infected cells. In cells constitutively expressing the E1B region of Ad2, Ad40 E1B mRNAs are detected at late times in infection, after the onset of DNA replication. We have determined the Ad40 E1B transcription map from RNA produced at late times in infected KB16 cells, using S1 nuclease, primer extension, PCR-cDNA analysis, and Northern blotting. E1B transcripts corresponding to Ad2 14 S, 22 S, and 9 S mRNAs were identified but no 13 S mRNA equivalent was detected, a pattern similar to that seen in the Ad12 transcription map. The coding potential for E1B 19K, 55K, and 15K proteins and for ppIX is retained in the Ad40 transcripts. In addition we find novel E1A-E1B cotranscript counterparts of the 14 S and 22 S mRNAs. These contain the first 40 codons of the E1A first exon linked to a site 4-5 nt downstream of the E1B cap site, retaining all the coding potential of the E1B mRNAs. No new open reading frames are created by the junction, and the E1A ORF terminates with one codon added after the junction. Each E1A-E1B cotranscript is present in abundance comparable to that of its authentic E1B counterpart. The E1A-E1B junction is unusual in that it does not conform to splice consensus sequences and thus may not be generated by a conventional splicing mechanism.