Technical Abstract: The cyclic lipopeptide profile of three Bacillus subtilis strains (AS 43.3, AS 43.4, and OH 131.1) was determined using mass spectroscopy. The strains are antagonists of Gibberella zeae and have been shown to be effective in reducing Fusarium head blight in wheat. Strains AS 43.3 and AS 43.4 produced the anti-fungal lipopeptide iturin A and to a much lesser extent fengycin during the stationary growth phase. All three strains transiently produced the lipopeptide surfactin during the exponential growth phase. Strain OH 131.1 only produced surfactin under these conditions. The amount of lipopeptide produced during culturing was estimated by measuring the surface tension of the culture media for each strain. The anti-fungal activity of culture supernatant was determined by the inhibition of G. zeae. Media from strains AS 43.3 and AS 43.4 demonstrated strong antibiosis of G. zeae, while strain OH 131.1 had no activity. These results suggest a different mechanism of antagonism for strain OH 131.1, relative to AS 43.3 and AS 43.4. The current results are interpreted in conjunction with previous bioassays to identify potential antagonism mechanisms.