Human beta-defensin 2 (hBD-2) and hBD-3 have potent fungicidal activity in the micromolar range. Although little is known about their mechanism of action against Candida species, some similarities to the antifungal mechanism of salivary peptide histatin 5 (Hst 5) seem to exist. Since hBD-2 and hBD-3 have been reported to cause direct disruption of target cell membranes, we compared the effects of hBD-2 and hBD-3 on Candida albicans membrane integrity. Incubation of calcein-loaded C. albicans cells with a dose of hBD-2 lethal for 90% of the strains tested (LD(90)) resulted in a maximal dye efflux of only 10.3% +/- 2.8% at 90 min, similar to that induced by Hst 5. In contrast, an LD(90) of hBD-3 more than doubled calcein release from cells yet did not result in more than 24% of total release, showing that neither peptide caused gross membrane damage. As for Hst 5, killing of C. albicans cells by hBD-2 and hBD-3 was salt sensitive; however, Ca(2+) and Mg(2+) inhibited hBD-2 but not hBD-3 fungicidal activity. Pretreatment of C. albicans cells with sodium azide resulted in significantly decreased ATP release and susceptibility of cells to hBD-2 and hBD-3. However, hBD-3 killing was partially restored at concentrations of > or =0.8 microM, showing energy-independent mechanisms at higher doses. C. glabrata resistance to Hst 5, hBD-2, and hBD-3 is not a result of loss of expression of cell wall Ssa proteins. The candidacidal effects of hBD-2-hBD-3 and Hst 5-hBD-2 were additive, while the index of interaction between Hst 5 and hBD-3 was 0.717 (P < 0.05). Thus, the candidacidal action of hBD-2 shows many similarities to that of Hst 5 in terms of salt sensitivity, ion selectivity, and energy requirements while hBD-3 exhibits biphasic concentration-dependent mechanisms of candidacidal action complementary to those of Hst 5.