We recently described the pre-steady state enzymatic binding kinetics of apurinic/apyrimidinic endonuclease (AP endo). In this report we describe the domain structure of the enzyme in solution determined by mild protease digestion in the presence and absence of substrate, product, and an efficient competitive inhibitor (HDP). AP endo is a 35.5-kDa protein with a high degree of homology to its prokaryotic counterpart, exonuclease III (Exo III), except for the amino terminus, which is lacking in the prokaryotic enzyme. The entire conserved region plus an additional 20 residues unique to the eukaryotic enzyme was inaccessible to trypsin and V8 protease, indicating that it forms a tight globular structure. In contrast, the amino-terminal 35 residues were readily accessible to all the proteases investigated, leading us to conclude that they associate poorly with the rest of the structure and constitute a highly fluid region. When AP endo was boiled with SDS and cooled prior to the addition of V8 protease, several acidic residues within the globular domain became protease-accessible, indicating rapid renaturation except along the nuclease fold with restoration of globular conformation for the carboxyl two-thirds of the molecule. Of all the proteases tested, only chymotrypsin was able to cleave internal to the globular portion without prior denaturation. Although AP endo cleaved with chymotrypsin retained full enzymatic activity, the activity was lost when the digested peptides were recovered after denaturation by heat and/or boiling in SDS, precipitation, and renaturation or when fragments were recovered from an SDS gel and renatured. Thus, the protein is probably held together strongly by noncovalent interactions that maintain enzymatic function after protease nicking. The three major chymotrypsin cleavage sites, Tyr-144, Leu-179, and Leu-205, became strikingly less accessible to protease digestion in the presence of abasic site-containing DNA. Since the three residues form a spherical triangle on the surface of the molecule on one side of the nuclease fold, there must be multiple means by which DNA containing an abasic site associates with the enzyme. The most likely explanation is that substrate and product, both of which were present during proteolysis, bind differently to the enzyme. Finally, the two cysteine residues thought to be involved in the redox reaction of AP endo with Jun protein were entirely inaccessible to proteolysis even after prolonged exposure of AP endo to reducing agents. Consequently, if AP endo plays a role in the physiological function of Jun, it must undergo major conformational changes in the process. Alternatively, the two cysteines could maintain an appropriate conformation such that other residues participate directly in the redox activity. Apurinic/apyrimidinic endonuclease (AP endo)1 is a critical enzyme in the repair of abasic sites in DNA. These sites can arise through spontaneous loss of a nucleobase, through the action of N-glycosyl hydrolases, or through oxidative damage of DNA (...