Nine samples of human ceruloplasmin [iron(II):oxygen oxidoreductase; EC 1.16.3.1] prepared by different procedures have been examined for heterogeneity; gel electrophoresis showed that seven contained a number of components with molecular weights ranging from 20,000 to 130,000, and two contained largely a single component of molecular weight 130,000. Digestion of a single-component preparation with plasmin produced fragments with molecular weights similar to those found in the multicomponent preparations. Amino-terminal analysis, peptide mapping, and amino acid analysis showed that plasmin digestion generated a fragment of 20,000 molecular weight, which corresponded to a component present in a multicomponent ceruloplasmin preparation. The 20,000 molecular weight fragment appears to correspond to the so-called alpha-subunit or L-chain of human ceruloplasmin. Chemical evidence is thus provided that ceruloplasmin is a single-chain protein and that the so-called subunits are fragments. The 20,000 molecular weight fragment contains a single cysteine; amino acid sequence studies have shown that the sequence in the vicinity of this residue is similar to that around the single cysteine residue in plant plastocyanins and bacterial azurins, which are small, blue, copper-containing proteins.