Next to almost all prokaryotic operons encoding peptide synthetases, which are involved in the nonribosomal synthesis of peptide antibiotics, distinct genes have been detected that encode proteins with strong sequence similarity to type II fatty acid thioesterases of vertebrate origin. Furthermore, sequence analysis of bacterial and fungal peptide synthetases has revealed a region at the C-terminal end of modules that are responsible for adding the last amino acid to the peptide antibiotics; that region also exhibits significant similarities to thioesterases. In order to investigate the function of these putative thioesterases in non-ribosomal peptide synthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis, srfA fragments encoding the thioesterase domain of the surfactin synthetase 3 and the thioesterase-like protein SrfA-TE were deleted. This led to a 97 and 84% reduction of the in vivo surfactin production, respectively. In the double mutant, however, no surfaction production was detectable. These findings demonstrate for the first time that the C-terminal thioesterase domains and the SrfA-TE protein are directly involved in nonribosomal peptide biosynthesis.