Electrostatic interactions in proteins can be dissected experimentally by determining the pKa values of their constituent ionizable amino acids. To complement previous studies of the glutamic acid and histidine residues in Bacillus circulans xylanase (BCX), we have used NMR methods to measure the pKa s of the seven aspartic acids and the C-terminus of this protein. The pKa s of these carboxyls are all less than the corresponding values observed with random coil polypeptides, indicating that their ionization contributes favorably to the stability of the folded enzyme. In general, the aspartic acids with the most reduced pKa s are those with limited exposure to the solvent and a high degree of conservation among homologous xylanases. Most dramatically, Asp 83 and Asp 101 have pKa s < 2 and thus remain deprotonated in native BCX under all conditions examined. Asp 83 is completely buried, forming a strong salt bridge with Arg 136. In contrast, Asp 101 is located on the surface of the protein, stabilized in the deprotonated form by an extensive network of hydrogen bonds involving an internal water molecule and the neutral side-chain and main-chain atoms of Ser 100 and Thr 145. These data provide a complete experimental database for theoretical studies of the ionization behavior of BCX under acidic conditions.