15Antimicrobial peptides (AMPs) have gained attention recently due to increasing antibiotic 16 resistance amongst pathogens. Most AMPs are cationic in nature and their preliminary 17 interactions with the negatively charged cell surface is mediated by electrostatic attraction. This 18 is followed by pore formation, which is either receptor-dependent or -independent and leads to 19 cell death. Typically, AMPs are characterized by their killing activity using bioactivity assays 20 to determine host range and degree of killing. However, cell surface binding is independent 21 from killing. Most of the studies performed to-date have attempted to quantify the peptide 22 binding using artificial membranes. Here, we use the narrow-spectrum class IIa bacteriocin 23 AMP pediocin PA-1 conjugated to a fluorescent dye as a probe to monitor cell surface binding. 24We developed a flow cytometry-based assay to quantify the strength of binding in target and 25 non-target species. Through our binding assays, we found a strong positive correlation between 26 cell surface charge and pediocin PA-1 binding. Interestingly, we also found inverse correlation 27 between zeta potential and pediocin PA-1 binding, the correlation coefficient for which 28 improved when only Gram-positives were considered. We also show the effect of the presence 29 of protein, salt, polycationic species, and other non-target species on the binding of pediocin 30PA-1 to the target organism. We conclude that the of presence of highly charged non-target 31 species, as well as solutes, can decrease the binding, and the apparent potency, of pediocin PA-32 1. Thus, these outcomes are highly significant to the use of pediocin PA-1 and related AMPs 33 in mixed microbial settings such as those found in the gut microbiota. 34 35