We purified a novel cysteine-rich antibiotic peptide, eNAP-2 (M(r), approximately 6,500), from acid extracts of equine neutrophils by sequential gel filtration and reversed-phase high-performance liquid chromatography and determined its partial N-terminal amino acid sequence. Although its cysteine motif distinguished eNAP-2 from all other currently known endogenous antibiotic peptides, including defensins and granulins, it showed substantial sequence similarity to WDNM1, a putative member of the four-disulfide-core protein family that also includes animal and human antiproteases, snake venom neurotoxins, and rat and mouse whey proteins. The antibacterial properties of eNAP-2 were tested against several equine uterine pathogens, namely, Streptococcus zooepidemicus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Killing of S. zooepidemicus was very efficient, as evidenced by a 94% decrease in numbers of CFU per milliliter after exposure to 100 micrograms of eNAP-2 per ml (approximately 15 microM) for 2 h. Exposure of E. coli and P. aeruginosa to 200 micrograms of eNAP-2 per ml for 2 h resulted in 90.2 and 77.6% reduction, respectively, in the numbers of CFU per milliliter. Bacteriostasis, without bactericidal activity, occurred after K. pneumoniae was incubated with 200 micrograms of eNAP-2 per ml. Additional studies will be required in other species and cell types to determine whether eNAP-2 is restricted to equine neutrophils or is the index member of a larger family of endogenous antibiotics.