Tryptic hydrolysis of whole casein was analyzed by high performance size exclusion chromatography (HPSEC) in combination with the degree of hydrolysis (DH). In terms of chromatograms obtained at different DH values and mass percentage calculated equations established by the normalization method, the complex process of enzymatic reaction and the molecular mass distribution of multiple hydrolysates were quantitatively characterized via 2-D plot. Based on the information of casein micelle structure, the possible reaction mechanism was deduced from a series of chromatographic results and experimental analysis. Taking into account the primary structure of whole casein and the target amino acid of trypsin, the distribution of theoretical peptides was accurately calculated by determining the split sites of complete enzymatic hydrolysis. According to the relationship between retention time and molecular mass, the corresponding chromatogram absorption peaks of active peptides in hydrolysates were identified, and caseinophosphopeptides (CPPs) sequence was also characterized. (C) 2003 Elsevier Science Ltd. All rights reserved.