Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk -casein composed of glycosylated and phosphorylated forms of different genetic variants. We have demonstrated that CMP has growth-inhibitory activity against the oral opportunistic pathogens Streptococcus mutans and Porphyromonas gingivalis and against Escherichia coli. CMP was fractionated using reversed-phase high-performance liquid chromatography (RP-HPLC), and each fraction was tested for activity against S. mutans in a 96-well-plate broth assay. Fractions were characterized by N-terminal sequence analysis and mass spectrometry. The active form of CMP was shown to be the nonglycosylated, phosphorylated -casein (residues 106 to 169) [-casein(106-169)], which we have designated kappacin. Endoproteinase Glu-C was used to hydrolyze CMP, and the generated peptides were separated using RP-HPLC and gel filtration-HPLC and then tested for activity against S. mutans. The peptide Ser(P)¹⁴⁹-casein-A(138-158) was the only peptide generated by endoproteinase Glu-C digestion that exhibited growth-inhibitory activity. Peptides corresponding to the sequences of the inhibitory peptide Ser(P)¹⁴⁹-casein-A(138-158) and its nonphosphorylated counterpart -casein-A(138-158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(P)¹⁴⁹ -casein-A(138-158) displayed growth-inhibitory activity against S. mutans (MIC, 59 µg/ml [26 µM]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.