The mechanism of action of antimicrobial peptides is still a matter of debate. The formation of ROS (reactive oxygen species) has been suggested to be the crucial step in the fungicidal mechanism of a number of antimicrobial peptides, including histatin 5 and lactoferrin-derived peptides. In the present study we have investigated the effects of histatin 5 and of a more amphipathic synthetic derivative, dhvar4, on the generation of ROS in the yeast Candida albicans, using dihydroethidium as an indicator for ROS. With both peptides, a substantial enhancement of fluorescence was observed. However, TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a cell-permeant ROS scavenger, did not have an inhibitory effect on killing or on the enhancement of fluorescence. Furthermore, antimycin and azide, which have been reported to induce ROS in vitro, were not able to enhance the dihydroethidium fluorescence, while chlorhexidine, a non-specific antiseptic agent, enhanced dihydroethidium fluorescence to the same extent as did the peptides. Fluorescence microscopy showed the fluorescence enhancement to be a consequence of the release of unbound preformed ethidium from the mitochondrial matrix within the cell. It is concluded that ROS do not play a role in the histatin 5-mediated killing of C. albicans.