The cationic peptide tritrpticin (VRRFPWWWPFLRR, Trp3) has a broad action spectrum, acting against Gram-positive and Gram-negative bacteria, as well as some fungi, while also displaying hemolytic activity. We have studied the behavior of Trp3 in planar lipid bilayers (or black lipid membrane – BLM) and were able to demonstrate its ion channel-like activity. Channel-like activity was observed in negatively charged azolectin BLM as a sudden appearance of discrete current fluctuations upon application of a constant voltage across the membrane. Trp3 formed large conductance channels (500–2000 pS) both at positive and negative potentials. In azolectin bilayers, the predominant ion-channel activity was characterized by very regular and discrete current steps (corresponding to openings) of uniform amplitude, which exhibited relatively long residence times (of the order of seconds). Occasionally, multiple conductance steps were observed, indicating the simultaneous presence of more than one open pore. In bilayers of zwitterionic diphytanoylphosphatidyl choline (DPhPC) Trp3 also showed ion-channel activity, but in a much less frequent and less prominent way. Studies of ion selectivity indicated that Trp3 forms a cation-selective channel. These results should contribute to the understanding of the molecular interactions and mechanism of action of Trp3 in lipid bilayers and biological membranes.