To isolate and characterize peptides from bovine colostrum with antimicrobial activity. Three peptides were purified from fresh colostrum by a range of chromatographic methods using antimicrobial activity against Escherichia coli DH5alpha to screen for the most active fractions. Two peptides, with antimicrobial activity, casecidin 17 and casecidin 15, were identical to sequences in the C-terminal of bovine beta-casein (YQEPVLGPVRGPFPIIV and YQEPVLGPVRGPFPI) and had corresponding molecular masses of 1881.00 and 1669.06 Da, respectively. The third peptide was the known peptide isracidin which has a mass of 2763.80 Da and sequence of RPKHPIKHQGLPQEVLNENLLRF. Casecidin 17 and casecidin 15 had identical minimal inhibition concentrations (MICs) against E. coli DPC6053 of 0.4 mg ml(-1). Structural modelling suggested amphiphilic structures having identical inhibitory and structural properties. The MIC value of isracidin against E. coli DPC6053 was 0.2 mg ml(-1). This study shows the presence of three antimicrobial peptides in colostrum which may contribute to a bioprotective role to limit pathogen contamination. Furthermore, the discovery of casecidin 17 and 15 may provide the basis for novel antimicrobial peptide design. This is the first study to characterize peptides with antimicrobial activity present in fresh bovine colostrum.