The adenoviral DNA binding protein (DBP) is a multifunctional protein involved in DNA replication and gene expression. In order to investigate the relation between structure and function of DBP, the amino acid sequences of the serotypes 5 and 12 (Ad5 and Ad12) have been compared. The amino acid sequence of Ad5 DBP was previously established by nucleotide sequence analysis of the Ad5 DBP gene (W. Kruijer, F. M. A. Van Schaik, and J. S. Sussenbach, Nucl. Acids Res. 9, 4439-4457, 1981). In this study the analysis of the Ad5 DBP gene and adjacent regions by determination of the sequence of the first leader in late DBP mRNA's and the splice point between the tripartite leader and the main body of the mRNA encoding the 100-kDa protein has been extended. The nucleotide sequence of the Ad12 DBP gene is also described. From the nucleotide sequence and RNA mapping data of Ad12 DBP mRNA's (I. Saito, J. Sato H. Handa, K. Shiraki, and H. Shimojo, Virology 114, 379-398, 1981) the complete Ad12 DBP amino acid sequence could be deduced. Ad12 DBP contains 484 amino acids and has an actual Mr of 54,992. It is 45 amino acids shorter than Ad5 DBP. Comparison of the Ad12 and Ad5 DBP amino acid sequences shows that several longer deletions are present in the N-terminal 125 amino acid residues of Ad12 DBP. In contrast, only a single amino acid deletion and insertion is found in the C-terminal 359 amino acids of Ad12 DBP. The N- and C-terminal domains of Ad12 and Ad5 DBP are 45 and 80% homologous, respectively. This suggests that both domains of DBP are subjected to different evolutionary pressures. Analysis of various Ad5 mutants with an altered DBP gene, has indicated that the C-terminal domain is involved in DNA replication and early gene expression, while the N-terminal domain has a role in late gene expression in monkey cells. These results are discussed in relation to the structure and function of adenovirus DBP.