Lysozyme, which is known to crystallize readily in the presence of many salts, has never been crystallized by salting out with ammonium sulfate. In the present study, lysozyme was first completely desalted by treatment with strong cation- (H(+) form) and anion- (OH(-) form) exchange resins. This leads to a protein solution with only H(+) and OH(-) as counterions, corresponding to its isoionic point. Addition of 2.5-3 molar equivalents of H(2)SO(4) to isoionic lysozyme decreases the pH value to 9-8 and allows crystallization to take place. The space group was found to be P4(3)2(1)2, similar to the classical lysozyme crystals grown in the presence of NaCl at pH 4.5, with unit-cell dimensions a = b = 78.9, c = 38.5 A. Tentative explanation of the sulfate/lysozyme interaction was addressed by mass spectrometry, and shows non-covalent binding of the ions on the protein.