Kappa-casein macropeptide (CMP) is one of the components of whey and is obtained as a by-product in cheesemaking. There has been increasing interest in research to find new uses of cheese industry by-products in order to improve their value and promote their use. Human platelet aggregation inhibitory activities of bovine, ovine, and caprine CMPs and their tryptic hydrolysates were studied. CMPs from the three species exhibited in vitro antithrombotic properties similar to the activity of the gamma-fibrinogen 400-411 peptide. Inhibitory activities increased following hydrolysis with trypsin. Active sequences were identified among the tryptic peptides by reversed-phase high-performance liquid chromatography with on-line mass spectrometry.