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Four new antibiotic peptaibols, named neoatroviridinsA (1), B (2), C (3), and D (4), have been isolated from theculture broth of Trichoderma atroviride. Their amino acidsequences were determined mainly by mass spectrometry incombination with NMR studies. The absolute stereochemistryof neoatroviridins was established as L by GC analysis of theiracid hydrolysates with derivatization, except for isovalinewhich was in D. Neoatroviridins, composed of 18 amino acidresidues, showed significant membrane-perturbing activityresponsible for their antibiotic action, which was comparableto that of alamethicin, a well-known 20-residue peptaibol.