The 18‐amino acid cytolytic lipodepsipeptide tolaasin, produced in culture by virulent strains of Pseudomonas tolaasii, is the causal agent of the brown blotch disease of the cultivated mushroom. Tolaasin has a sequence of D‐amino acids in its N‐terminal region, then alternates L‐ and D‐amino acids, and bears a C‐terminal lactone macrocycle composed of 5‐residues. The solution structure of tolaasin in sodium dodecyl sulfate was studied by 2D‐NMR spectroscopy and molecular dynamics simulated annealing calculations. Tolaasin forms an amphipathic left‐handed α‐helix in the regionDPro2‐DalloThr14 comprising the sequence of seven D‐amino acids and the adjacent L‐D‐L‐D‐D‐region. To the best of our knowledge, this is the first recognized example of a left‐handed α‐helix including both D‐ and L‐amino acids. The lactone macrocycle adopts a “boat‐like” conformation and is shifted from the helical axis as to form a “golf‐club” overall conformation. These structural features will be of importance in understanding, and preventing, tolaasin's role in the bacterial colonization of the host plant, and its toxic action on cells. Furthermore, the observed antimicrobial activity together with the potential resistance to enzymatic degradation and the increased antigenicity (both due to the presence of L‐ and D‐amino acids) strongly suggests for tolaasin a potential role as a template model for the design of new therapeutic antibacterial molecules. Proteins 2003;52:534–543. © 2003 Wiley‐Liss, Inc.