A 606 base pair segment of adenovirus type 2 DNA which includes the gene for polypeptide IX and its flanking regions has been sequenced. Comparison with the sequence of a cDNA copy of the 9S mRNA for polypeptide IX and direct analysis of its capped oligonucleotide show that the gene and its mRNA are colinear. The 9S mRNA has a length of 485 nucleotides, excluding the poly(A) tract and the m7G of the cap, and contains one single open translational reading frame which is sufficiently long to encode polypeptide IX. This reading frame contains three AUG codons, the first of which is used for initiation of protein synthesis. The mRNA has a 5' noncoding region of 24 nucleotides, excluding the m5G of the cap, and encodes a polypeptide which is 139 amino acids long and unusually rich in serine and alanine. The sequence TATATAA is found 25 nucleotides upstream from the cap site. The polyadenylation site has been identified but cannot be located unambiguously because of the presence of two A residues in the DNA sequence at this position. The 3' noncoding region is 41--43 nucleotides long and contains the hexanucleotide sequence AAUAAA. The mRNA for polypeptide IVa2 is encoded adjacent to that for polypeptide IX and is transcribed in the opposite direction. Sequence analysis revealed an overlap of 11--16 nucleotides, depending on the precise location of the polyadenylation sites, between 3' ends of these mRNAs.