Peptides from phosphorylated and dephosphorylated casein hydrolysates were fractionated on a TSK G2000SW size-exclusion column. The fractionated peptides were separated by reversed-phase high-performance liquid chromatography on a C 18 column using aqueous trifluoroacetic acid as the mobile phase and acetonitrile as the mobile phase modifier in the linear gradient elution system. The separation of the dephosphorylated and phosphorylated hydrolysates gave 213 and 187 peptides, respectively, of which 116 and 99, respectively, were reported. A study of their composition and retention times verified that the peptide separation mechanism includes ionic interactions, hydrogen bonding and peptide characteristics, in addition to overall peptide hydrophobicity.