The fungus Trichoderma virens is a ubiquitous soil saprophyte that has been applied as a biological control agent to protect plants from fungal pathogens. One mechanism of biocontrol is mycoparasitism, and T. virens produces antifungal compounds to assist in killing its fungal targets. Peptide synthetases produce a wide variety of peptide secondary metabolites in bacteria and fungi. Many of these are known to possess antibiotic activities. Peptaibols form a class of antibiotics known for their high alpha-aminoisobutyric acid content and their synthesis as a mixture of isoforms ranging from 7 to 20 amino acids in length. Here we report preliminary characterization of a 62.8-kb continuous open reading frame encoding a peptaibol synthetase from T. virens. The predicted protein structure consists of 18 peptide synthetase modules with additional modifying domains at the N- and C-termini. T. virens was shown to produce a mixture of peptaibols, with the largest peptides being 18 residues. Mutation of the gene eliminated production of all peptaibol isoforms. Identification of the gene responsible for peptaibol production will facilitate studies of the structure and function of peptaibol antibiotics and their contribution to biocontrol activity.