An initial study was designed validate an in vitro antimicrobial assay and evaluate the activity of natural antimicrobial animal proteins/peptides. A radial diffusion assay was utilized. By regressing known concentrations of tested compound against their respective clearance zones, an equation was developed to determine the minimum inhibitory concentrations (MIC) for polymyxin B (control antibiotic) which were 0.76, 0.76, and 0.90 mug/mL for Escherichia coli, Escherichia coli (nalidixic acid-resistant), and Staphylococcus aureus, respectively. The intra- and inter-assay variations were 0.18 and 0.2%, respectively. Lactoferrin, lactoferricin B, hen egg lysozyme, and alpha-lactalbumin LDT2 were determined in vitro to kill bacteria. Each of the tested proteins/peptides was active against a nalidixic acid-resistant strain of E. coli. The antimicrobial activity of each protein/peptide in animal digesta fluid was 130 to 300% greater than that in the acetic acid media. Lactoferrin activity was decreased (P < .07) when exposed to zinc, iron, magnesium, calcium, sodium, and potassium. Lysozyme had an increase (P < .09) in antimicrobial activity when exposed to zinc, magnesium, and calcium. A decrease in pH from 6.5 to 2 resulted in a loss of antimicrobial activity of 65% for lactoferrin, lactoferricin, and lysozyme. Therefore, the enhanced antimicrobial activity of the proteins/peptides in the presence of the digesta fluid does not seem to be affected by pH 44 or mineral concentration.;A second study was performed to evaluate the efficacy of orally ingested natural proteins/peptides contained in digesta contents to kill antibiotic-susceptible and -resistant bacteria, in vitro. The seven treatments consisted of a control (no antimicrobial agent supplemented), lactoferrin, lactoferricin, lysozyme, neutrophil peptides, purothionin, or polymyxin B (antibiotic). Weanling Sprague-Dawley rats were self-fed their respective experimental diets for 3 days. Following oral ingestion, each protein/peptide exhibited antimicrobial activity in the small and large intestine digesta (P < .06) and against Gramnegative and Gram-positive bacteria (P < .11) (both antibiotic-susceptible and -resistant strains, P < .10). The antibiotic polymyxin B also effectively killed in vivo each bacterium except antibiotic-resistant Staphylococcus aureus, while the control diet exhibited no antimicrobial activity. Based on these results, ingested natural proteins/peptides retain antimicrobial activity in monogastric animals throughout the gastrointestinal tract and effectively kill both antibiotic-susceptible and -resistant bacteria.