Objective--To evaluate antimicrobial activity of bovine bactericidal permeability-increasing protein (bBPI)-derived synthetic peptides against mastitis-causing gram-negative bacteria. Sample Population--Bacterial isolates from the milk of cows with clinical mastitis. Procedures--3 peptides were synthesized with sequences corresponding to amino acids 65 to 99 (bBPI6599) or 142 to 169 (bBPI142169) or the combination of amino acids 90 to 99 and 148 to 161 (bBPI9099,148161) of bBPI. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of these peptides against bacterial isolates from cows with mastitis were determined by use of a standardized broth microdilution assay. The ability of these peptides to retain their antimicrobial activity in serum and milk was also evaluated. Finally, bacterial lipopolysaccharide (LPS)-neutralizing activity of these peptides was assayed with the Limulus amebocyte lysate test. Results--Of the 3 peptides tested, bBPI9099,148161 had the widest spectrum of antimicrobial activity, with MIC and MBC values ranging from 16 to 64 Mg/mL against Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp and from 64 to 128 Mg/mL against Pseudomonas aeruginosa. None of the peptides had any growth-inhibitory effect on Serratia marcescens. The antimicrobial activity of bBPI9099,148161 was inhibited in milk, but preserved in serum. Finally, bBPI142169 and bBPI9099,148161 completely neutralized LPS. Conclusions and Clinical Relevance--bBPI9099,148161 is a potent neutralizer of the highly proinflammatory molecule bacterial LPS and has antimicrobial activity against a variety of gram-negative bacteria. The ability of bBPI9099,148161 to retain antimicrobial activity in serum suggests a potential therapeutic application for this peptide in the management of gram-negative septicemia.