Salivary histatins (Hsts) are potent candidacidal proteins that induce a nonlytic form of cell death in Candida albicans accompanied by loss of mean cell volume, cell cycle arrest, and elevation of intracellular levels of reactive oxygen species (ROS). Since these phenotypes are often markers of programmed cell death and apoptosis, we investigated whether other classical markers of apoptosis, including generation of intracellular ROS and protein carbonyl groups, chromosomal fragmentation (laddering), and cytochrome c release, are found in Hst 5-mediated cell death. Increased intracellular levels of ROS in C. albicans were detected in cells both following exogenous application of Hst 5 and following intracellular expression of Hst 5. However, Western blot analysis failed to detect specifically increased protein carbonylation in Hst 5-treated cells. There was no evidence of chromosomal laddering and no cytochrome c release was observed following treatment of C. albicans mitochondria with Hst 5. Superoxide dismutase enzymes of C. albicans and Saccharomyces cerevisiae provide essential protection against oxidative stress; therefore, we tested whether SOD mutants have increased susceptibility to Hst 5, as expected if ROS mediate fungicidal effects. Cell survival of S. cerevisiae SOD1/SOD2 mutants and C. albicans SOD1 mutants following Hst 5 treatment (31 micro M) was indistinguishable from the survival of wild-type cells treated with Hst 5. We conclude that ROS may not play a direct role in fungicidal activity and that Hst 5 does not initiate apoptosis or programmed cell death pathways.