Antibacterial peptides were purified from porcine neutrophil granules collected from healthy pigs. Granule proteins, extracted with 0.2 mol/L sodium acetate were subjected to ion-exchange chromatography and five peaks (designated A to E) were detected. Individual porcine neutrophil granule proteins were shown to inhibit the growth of target organisms Escherichia coli and Staphylococcus aureus. The antimicrobial activity was shown to be concentration and time dependent. Peak D showed strong antimicrobial activity against S. aureus and peak C (with a greater number of eluted proteins) was shown to be active against both S. aureus and E. coli. One of the peptides was purified further by reverse-phase HPLC from peak fraction C. The MW of this peptide was approximately 5500 Da as determined by SDS-PAGE and mass spectral analysis and was active against both E. coli and S. aureus in vitro sustaining a > 90% decrease, respectively, in CFU after a 2 h exposure with 50 micrograms of this peptide. Amino acid analysis showed the peptide was rich in aspartate/aspartic acid, glutamine/glutamic acid, proline, arginine and threonine. The antimicrobial activity of this peptide and other novel proteins in porcine neutrophilic granules demonstrates the probable role of these proteins and peptides in host defence of porcine neutrophils against bacterial infection.