Oral epithelial cell-derived human beta-defensins-1, -2, and -3 participate in innate immune responses against Candida. We hypothesized that these peptides utilize several mechanisms for protection. Recombinant hBD-1 and -2 were produced with the use of an insect cell/baculovirus expression system, while rhBD-3 was expressed as a fusion protein in E. coli. RhBD-2 and -3 were more effective at killing the candidal species at low micromolar concentrations than was rhBD-1, except for C. glabrata. While this species was relatively resistant to rhBD fungicidal activity, its adherence to oral epithelial cells was strain-specifically inhibited by the rhBDs. C. albicans hyphae were important in regulating hBD2 and -3 mRNA expression in primary human oral epithelial cells. Confocal microscopy of rhBD-2-challenged C. albicans suggests disruption of the fungal membrane. Results support the hypothesis that hBDs control fungal colonization through hyphal induction, direct fungicidal activity, and inhibition of candidal adherence.