Cationic lipodepsipeptides from Pseudomonas spp. have been characterized for their structural and antimicrobial properties. In the present study, the structure of a novel lipodepsipeptide, cormycin A, produced in culture by the tomato pathogen Pseudomonas corrugata was elucidated by combined protein chemistry, mass spectrometry and two-dimensional NMR procedures. Its peptide moiety corresponds to L-Ser-D-Orn-L-Asn-D-Hse-L-His-L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-Cl) [where Orn represents ornithine, Hse is homoserine, aThr is allo-threonine, Z-Dhb is 2,3-dehydro-2-aminobutanoic acid, Asp(3-OH) is 3-hydroxyaspartic acid and Thr(4-Cl) is 4-chlorothreonine], with the terminal carboxy group closing a macrocyclic ring with the hydroxy group of the N-terminal serine residue. This is, in turn, N-acylated by 3,4-dihydroxy-esadecanoate. In aqueous solution, cormycin A showed a rather compact structure, being derived from an inward orientation of some amino acid side chains and from the 'hairpin-bent' conformation of the lipid, due to inter-residue interactions involving its terminal part. Cormycin was significantly more active than the other lipodepsipeptides from Pseudomonas spp., as demonstrated by phytotoxicity and antibiosis assays, as well as by red-blood-cell lysis. Differences in biological activity were putatively ascribed to its weak positive net charge at neutral pH. Planar lipid membrane experiments showed step-like current transitions, suggesting that cormycin is able to form pores. This ability was strongly influenced by the phospholipid composition of the membrane and, in particular, by the presence of sterols. All of these findings suggest that cormycin derivatives could find promising applications, either as antifungal compounds for topical use or as post-harvest biocontrol agents.