Ceruloplasmin (EC 1.16.3.1) is a copper-containing alpha 2-glycoprotein and a member of the acute phase reactant family. Fragmentation of ceruloplasmin during purification and storage has hampered studies of its structure, but it has been shown to be a 132-kDa monomer. Combining two published chromatographic steps with additional gel filtration and fast protein liquid chromatography (FPLC) steps, we now report a procedure that yields a highly purified and nonlabile protein. Human plasma was subjected to QAE-Sephadex A-50 chromatography, precipitated with ammonium sulfate, and chromatographed on a hydroxyapatite column. The resulting protein was > 95% pure but highly unstable as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; at 37 degrees C the 132-kDa protein disappeared completely within 48 h. Further purification of ceruloplasmin by Sephadex G-50 chromatography and Mono Q FPLC yielded a protein that was essentially pure by multiple criteria and completely stable even after incubation at 37 degrees C for 4 weeks. When purified ceruloplasmin was reconstituted with fractions eluted from the Sephadex G-50 column, a single fraction induced proteolytic degradation. The degradation of ceruloplasmin by this fraction was inhibited by EDTA and 1,10-phenanthroline, indicating that a plasma metalloproteinase is responsible for degradation of ceruloplasmin.