Putative phosphopeptides produced from enzymehydrolysis of phosvitin were identified and characterizedusing MALDI-TOF/MS. Phosvitin was heat-pretreated andthen hydrolyzed using pepsin, thermolysin, and trypsin attheir optimal pH and temperature conditions with or withoutpartial dephosphorylation. Pepsin and thermolysin were noteffective in producing phosphopeptides, but trypsinhydrolysis produced many peptides from phosvitin: 12peptides, 10 of which were phosphopeptides, were identifiedfrom the trypsin hydrolysate. Twelve peptides were alsoidentified from the trypsin hydrolysate of partiallydephosphorylated phosvitin, but the number of phosphategroups remaining with the peptides was much smaller thanthose from the trypsin hydrolysate of intact phosvitin. Thissuggested that the phosphopeptides produced from thepartially dephosphorylated phosvitin lost most of theirphosphate groups during the dephosphorylation step.Therefore, partial dephosphorylation of phosvitin beforetrypsin hydrolysis may not be always recommendable inproducing functional phosphopeptides if the phosphategroups play important roles for their functionalities.