There is a general agreement that the experimentally determined molecular weight (MW) of caseinomacropeptide (CMP) is greater than the theoretical MW. Some studies suggest that this is due to a pH-dependent aggregation of monomeric CMP. How this aggregation is influenced by pH is not understood. This study was carried out to study the nature of CMP aggregates and to clarify which conditions affect aggregation of CMP. The apparent MW of CMP at different pH values was determined using size-exclusion chromatography. Caseinomacropeptide was further characterized by immunochemical analysis, sodium dodecyl sulfate-PAGE, N-terminal sequencing, and mass spectrometry. The hydrophobicity of CMP was studied by means of 1-anilinonaphthalene-8-sulfonic acid binding experiments. Four CMP products prepared by different methods were studied: CMP produced by enzymatic (chymosin or pepsin) hydrolysis of κ-casein (CN), and 2 commercial CMP products. Both commercial products and CMP resulting from chymosin-hydrolysis of κ-CN (at pH 6.6) had elution volumes with a MW corresponding to 35 kDA at pH 8.0 and 3.4. Caseinomacropeptide prepared from pepsin-hydrolysis of κ-CN (at pH 2.5) eluted as multiple peaks with apparent MW of 35, 18, and 9 kDa, again independently of pH. Hydrolysis of κ-CN with chymosin or pepsin at different pH values (pH 2.5, 3.4, and 6.6) produced differently sized aggregates of CMP, largely depending on the pH of the hydrolysis. These results indicate that, whereas CMP molecules are irreversibly associated, CMP in κ-CN may associate reversibly in a pH-dependent manner. We suggest that interactions between para-κ-CN parts of the κ-CN molecules may be a requisite for the pH-dependent dissociation/association.