Plants have developed a variety of mechanisms to cope with abiotic and biotic stresses. In a previous subcellular localization study of hydrogen peroxide-responsive proteins, two peptides with an unknown function (designated ARACIN1 and ARACIN2) have been identified. These peptides are structurally very similar, but are transcriptionally differentially regulated during abiotic stresses, during Botrytis cinerea infection or after benzothiadiazole and methyl jasmonate treatments. In Arabidopsis (Arabidopsis thaliana), these paralogous genes are positioned in tandem within a cluster of pathogen defense-related genes. Both ARACINs are small, cationic and hydrophobic peptides, known characteristics for antimicrobial peptides. Their genes are expressed in peripheral cell layers prone to pathogen entry and are lineage-specific to the Brassicaceae family. In vitro bioassays demonstrated that both ARACIN peptides have a direct antifungal effect against the agronomically and economically important necrotrophic fungi, Botrytis cinerea, Alternaria brassicicola, Fusarium graminearum, Sclerotinia sclerotiorum, and the yeast Saccharomyces cerevisiae. In addition, transgenic Arabidopsis plants that ectopically express ARACIN1 are protected better against infections with both Botrytis cinerea and Alternaria brassicicola. Therefore, we can conclude that both ARACINs act as antimicrobial peptides.