The principal active component of tolaasin, the Pseudomonas tolaasii toxin, which is responsible for brown blotch disease of mushrooms, is shown to be a lipodepsipeptide of M, 1985. The structure determined is 8-hydroxyoctanoyl-ABut-~Pro-~Ser-D-Leu-~Val-D-Ser-~Leu-D-Val-~-Va~-D-Gln-~-Leu-D-Val-ABut-D-all~Thr-~-I~~~-H~-~Dab~-L~ (cyclized via lactone formation between D-affeThr and the C-terminus) by a combination of fast atom bombardment mass spectrometry (FABMS), 'H NMR, automated sequencing, and chiral gas chromatography. A minor component of the toxin (M, 1941) has a related structure in which the homoserine residue is substituted by glycine. In addition, the sequence of a modified form of the toxin, produced by a genetically engineered strain of P. tofaasii, is characterized.