Early region 4 (E4) of mouse adenovirus type 1 was analyzed by Northern blotting, cDNA sequencing, and S1 nuclease protection and primer extension assays. The transcription map of this region was dissimilar to the consensus human adenovirus E4 transcription map in which all transcripts have identical 5' and 3'-terminal sequences. Seven classes of mouse adenovirus type 1 mRNAs were identified; all shared the same 3' end. Three classes of unspliced mRNAs differed at their 5' start sites, two classes of spliced transcripts differed in the locations of their splice acceptors, and two classes of spliced messages differed in their splice donors and acceptors. From the structure of the various transcripts, translational products were predicted. In addition to a predicted polypeptide with similarity to the human adenovirus 2 E4 34K protein previously identified (A. O. Ball, C. W. Beard, P. Villegas, and K. R. Spindler, 1991, Virology 180, 257-265), two open reading frames with similarity to human adenovirus 2 E4 open reading frames 2 and 3 were found.