Herein, we report on the mutational analysis of a 70-amino acid segment (region 1, residues 438-508) of family B DNA polymerase from the thermoacidophilic archaeon Sulfolobus solfataricus (Sso DNA pol). Region 1, which lies between the Exo III sequence and the similarity motif D- -SLYP, connects the exonuclease and polymerase domains of Sso DNA pol. Two C-terminally deleted forms of the enzyme, proteins N438 (residues 1-438) and N508 (residues 1-508), were overproduced in the recombinant form and biochemically characterized. They contain the three evolutionarily conserved Exo motifs, but differ in the extent of the C-terminal deletion, since only N508 includes region 1. Both have been found to retain a Mn2+-dependent 3'-5' exonuclease activity, whose thermal stability appears to be increased in comparison to that of the full-sized enzyme. Assays for processive 3'-5' exonuclease activity, carried out with the heparin trap method on a 24-base oligonucleotide, have revealed that protein N508, as well as the full-length Sso DNA pol, retains a level of processivity of the degradative function substantially higher than that for protein N438. In addition, six site-specific mutations have been introduced at the highly conserved Y-GG/A motif, which has been found within Sso DNA pol region 1. All mutant proteins (Lys491Ile, Tyr495Ser, Lys496Ile, Gly497Ala, and Ala498Val) display increased processivity of their 3'-5' exonuclease activity, with the exception of protein Tyr495Phe. By a steady-state kinetic analysis of the exonucleolytic reaction on a 24-base oligonucleotide, the above site-specific mutations have been found to affect Km values consistently with the observed differences in the processivity values, whereas the effect on the kcat values seems to be less important. The results from this mutational analysis indicate that region 1 is involved in determining the processivity of the proofreading function, directly interacting with the nucleic acid substrate.