Three new enniatins, B2, B3, and B4, were isolated from the hexane extracts of liquid cultures of Fusarium acuminatum MRC 3308, in addition to the four known enniatins, A, AI, B, and B1. The different enniatins were purified by reversed-phase semipreparative HPLC and could be detected directly in the extracts by FAB-MS or GUMS. Enniatin B2 was characterized as N-demethylenniatin B by its NMR, IR, and mass spectra. Enniatin Bs was tentatively assigned the structure NJV-didemethylenniatin B on the basis of FAB-MS evidence. Enniatin Bq, an isomer of enniatin B1, was shown by NMR to contain an N-methylleucine moiety. This is the first account of N-demethylenniatins (Bz and B3) being produced by fungi. Most of the tested strains of F. acuminatum (3 of 5 strains) and Fusarium compactum (8 of 10 strains) from different geographic origins produced one or more of the above enniatins.