Ostricacin-1 and ostricacin-2 (Osp-1 and Osp-2) were beta-defensins antimicrobial peptides that were purified from ostrich leukocytes using a cation-exchange column and a semi-prep RP-HPLC column. Both ostricacins were subjected to increased concentrations of monovalent cations (K+ and Na+ and divalent cations (Ca2+ and Mg2+ in order to investigate the effect of cations on the activity of these ostricacins on Gram-negative bacteria and Gram-positive bacteria. The radial diffusion assay method showed that both ostricacins were sensitive to the presence of cations. The divalent cations showed more antagonized effect on the activity against Gram-negative bacteria than the monovalent cations, as the ostricacins lost ability to inhibit bacterial growth at very low concentration (5 mM). When viewed in the context of other defensins activity, our data support a hypothesis that defensins' overall net positive charge determine the sensitivity to cations.