Harzianin HB I is a minor component of the peptaibol mixture biosynthesized by a Uruguayan strain of Trichoderma harzianum. It was isolated from the particularly complex mixture of 14-residue harzianins HC by repetitive semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). Its sequence was elucidated by liquid secondary ion mass spectrometry (LSIMS) and two-dimensional 1H NMR experiments. This 11-residue peptaibol is an analogue of the 14-residue peptaibol harzianin HC IX, resulting from the deletion of the tripeptide Aib-Pro-Ala. The CD and NMR data (NOE data and amide proton temperature coefficients) are similar to those of HC IX, suggesting a right-handed helix-type conformation for the two peptides. Harzianin HB I was synthesized by the solution-phase method using BOP as coupling reagent. It was shown to modify slightly the permeability of liposomes and induce voltage-gated macroscopic conductance in planar lipid bilayers. Its membrane properties examined on liposomes were compared with those of other known peptaibols.