The aim of this study was to assess the in vitro antimicrobial activity of Pep(27) and its mode of action. The results indicated Pep27 exhibited antibacterial activities against pathogenic Gram-positive and Gram-negative bacteria without hemolytic effect on human erythrocytes, but it did not exhibit antifungal activity. To understand the mode of action of Pep(27) the cellular distribution of the peptide was investigated. Flow cytometry analysis exhibited Pep(27) penetrated the bacterial membrane by an en energy- independent pathway without any damage to the membrane when examined using liposome and membrane probe. After penetration into the bacterial cells, Pep(27) was not affected by macromolecular synthesis, but activated protein phosphatase activity in dose ranges of 10-15 mu M and time range of 5-10 min in case of Staphylococcus epidermidis and Pseudomonas aeruginosa, respectively. These results demonstrated the antibacterial activities of Pep 27 are fundamentally attributable to a physiological change by activated phosphatase activity. (C) 2007 Elsevier Inc. All rights reserved.