The Protein Identification Resource (PIR) protein sequence data bank was searched for sequence similarity between known proteins and human DNA polymerase beta (Pol beta) or human terminal deoxynucleotidyltransferase (TdT). Pol beta and TdT were found to exhibit amino acid sequence similarity only with each other and not with any other of the 4750 entries in release 12.0 of the PIR data bank. Optimal amino acid sequence alignment of the entire 39-kDa Pol beta polypeptide with the C-terminal two thirds of TdT revealed 24% identical aa residues and 21% conservative aa substitutions. The Monte Carlo score of 12.6 for the entire aligned sequences indicates highly significant aa sequence homology. The hydropathicity profiles of the aligned aa sequences were remarkably similar throughout, suggesting structural similarity of the polypeptides. The most significant regions of homology are aa residues 39-224 and 311-333 of Pol beta vs. aa residues 191-374 and 484-506 of TdT. In addition, weaker homology was seen between a large portion of the 'nonessential' N-terminal end of TdT (aa residues 33-130) and the first region of strong homology between the two proteins (aa residues 31-128 of Pol beta and aa residues 183-280 of TdT), suggestive of genetic duplication within the ancestral gene. On the basis of nucleotide differences between conserved regions of Pol beta and TdT genes (aligned according to optimally aligned aa sequences) it was estimated that Pol beta and TdT diverged on the order of 250 million years ago, corresponding roughly to a time before radiation of mammals and birds.