Family B DNA polymerase from the thermoacidophilic archaeon Sulfolobus solfataricus (Sso DNA pol) is a monomer of about 100 kDa with two associated catalytic functions: 3'-5' exonuclease and DNA polymerase activities. The structure of this enzyme in the free and DNA-bound states was probed by limited proteolysis and fluorescence spectroscopy measurements. The results of partial trypsin proteolysis experiments on the recombinant Sso DNA pol pinpointed three major sites of protease sensitivity: near the N-terminus, within the center, and near the C-terminal end of the polypeptide chain. When partial trypsin digestion was carried out in the presence of either activated calf thymus DNA or primed M13mp18 single-stranded DNA, changes in cleavage pattern and in susceptibility to protease were detected. This phenomenon was dependent on the nucleic acid concentration and suggested the occurrence of DNA-induced conformational changes. These were also probed by steady-state fluorescence spectroscopy measurements using acrylamide as a quencher. Fine mapping of the DNA-specific cleavage sites allowed us to precisely locate the protein subdomains which were affected by these structural changes. Importantly, a specific proteolytic fragment of about 8 kDa was recovered after partial digestion of Sso DNA pol only in the presence of nucleic acid ligands. It was found to start at residues 392-394 and to span the protease-hypersensitive central region of the polypeptide chain. Its involvement in critical polymerase functions, such as substrate binding and/or enzyme processivity, was discussed. In addition, we found that controlled trypsin digestion of Sso DNA pol did not inactivate either polymerase or 3'-5' exonuclease activity concomitantly with the disappearance of full-sized enzyme. Activity gel analysis revealed that proteolytic products corresponding to the amino- and carboxyl-terminal halves of the enzyme retained 3'-5' exonuclease and DNA polymerase activity, respectively. These results are in line with the model of modular organization proposed for Sso DNA pol in a previous report.