PAF26 is a synthetic fungicidal hexapeptide with cell-penetration properties and non-lytic mode of action. We demonstrate herein the endogenous accumulation of reactive oxygen species (ROS) and nitric oxide (NO) in the model fungus Saccharomyces cerevisiae treated with PAF26. However, the S. cerevisiae deletion mutant of YAP1 - the major inductor of defense to oxidative stress - did not show high sensitivity to PAF26 but rather increased resistance, and its ROS accumulation did not differ from that of the parental strain. Cross-protection experiments suggest that the oxidant H2O2 and PAF26 kill yeast through different pathways. Overall, the data indicate that ROS are not the primary antifungal mechanism of the peptide. On the contrary, the PAF26-induced intracellular production of NO was blocked in two distinct resistant mutants: the above mentioned Delta yap1, which had the induction of NO disrupted, and the previously reported Delta arg1 from the biosynthetic pathway of arginine, which has reduced basal NO levels. The NO synthase inhibitor L-NAME partially restored yeast growth in the presence of PAF26. These findings correlate antifungal activity of PAF26 with NO production and provide a plausible explanation for the resistance phenotype of Delta arg1 through its involvement in NO biosynthesis. (C) 2011 Elsevier Inc. All rights reserved.