The enzymatic formation of the cyclodepsipeptide beauvericin [with insecticidal property] was demonstrated in cell-free extracts from Beauveria bassiana [an insect pathogen]. In analogy to the enniatin synthetase system formation of beuvericin is strictly dependent on the presence of the constituent amino and hydroxy acid, S-adenosylmethionine, and ATP/Mg2+. Synthesizing activity could be enriched about 12-fold by fractional ammonium sulfate precipitation. Besides the enniatin synthetase system this represents another example of the cell-free synthesis of a depsipeptide from eucaryotic origin.