Polymorphonuclear leukocytes (PMN) contribute to epithelial injury at sites of inflammation, but their mechanisms of action are incompletely understood. PMN can injure target tissues by oxidative and nonoxidative mechanisms. Included in the nonoxidative mechanisms are defensins (DEF), small (3.5 to 4.0 kD), arginine- and cysteine-rich polypeptides. DEF are bactericidal, fungicidal, viricidal, and tumoricidal, but their ability to contribute to inflammatory injury has not been extensively evaluated. One marker of inflammatory injury is disrupted epithelial barrier integrity. Using Madin-Darby canine kidney (MDCK) epithelial monolayers, we measured the effect of both human and rabbit DEF on barrier integrity using mannitol permeability (P_mann) and transepithelial electrical resistance (R_t). Human DEF (HNP1–3, 2:2:1 molar ratio) increased P_mann in a time- and concentration-dependent manner and R_t fell progressively over a 48-h period after exposure of monolayers to HNP1–3. Rabbit DEF peptide 1 (NP-1) also increased P_mann, but rabbit peptide 5 (NP-5) had no effect on P_mann. To investigate the role of charge, HNP1–3 was added to the monolayers with the polyanions heparin or sulfated dextran. Heparin and sulfated dextran only partially inhibited the increase in P_mann. Fetal bovine serum (FBS), however, completely inhibited the effect of HNP1–3, but this protection was only partially explained by the anionic protein, albumin. The FBS protection was time dependent and was present when FBS was added up to 16 h after exposure to HNP1–3. While both HNP1–3 and NP-1 increased epithelial permeability, neither were cytolytic to MDCK cells as measured by lactate dehydrogenase release. These studies demonstrate that HNP1–3 and NP-1 alter the barrier integrity of a cultured epithelium without causing cytolytic injury and expand the possible contribution of DEF to inflammatory injury.