Functions of the terminal domains of the family D DNA polymerase from Pyrococcus horikoshii (PolDPho) were analyzed by making and characterizing various truncated proteins. Based on a co-expression vector developed previously (Shen et al. (2001), J. Biol. Chem. 276: 27376-27383), twenty-five vectors for terminal truncated proteins were constructed. The expressed proteins were characterized in terms of thermo-stability, subunit interaction, and polymerization and 3'-5' exonuclease activities. The carboxyl terminal (1255-1332) of the large subunit (DP2Pho) and two regions, the 201-260 and 599-622 of the small subunit (DP1Pho) were found to be critical for the complex formation, and probable subunit interaction of PolDPho. The amino terminal (1-300) of DP2Pho is essential for the folding of PolDPho and is likely the oligomerization domain of PolDPho. A short region at the extreme C-terminal of DP2Pho (from 1385 to 1434) and the N-terminal of DP1Pho (1-200), which forms a stable protein, are not absolutely necessary for either polymerization or the 3'-5' exonuclease activity. We identified a possible regulatory role of DP1Pho (1-200) for the 3'-5' exonuclease. Deletion of DP1Pho (1-200) increased the exonuclease and DNA binding activities of PolDPho. Adding DP1Pho (1-200) to the truncated protein suppressed the elevated exonuclease activity. We also constructed and analyzed three internal deletion mutants and two site-directed mutants in the region of the putative zinc finger motif (cystein cluster II) of DP2Pho at the C-terminal. We found that the internal region of the zinc finger motif is critical for the 3'-5' exonuclease, but is dispensable for the DNA polymerization.