Several Fusarium strains produce the cyclohexadepsipeptide enniatin, a host-nonspecific phytotoxin. Enniatins are synthesized by the 347-kDa multifunctional enzyme enniatin synthetase. In the present study, 36 Fusarium strains derived from a wide range of host plants were characterized with respect to enniatin production in different media. Thirteen of these strains produced enniatins on one or more of these media. To determine whether enniatin production affected virulence, an assay on potato tuber tissue was performed. Seven enniatin-producing and 16 nonproducing strains induced necrosis of potato tuber tissue, so that enniatin synthesis is not essential for the infection of potato tuber tissue. The application of a mixture of enniatins to slices of potato tuber, however, caused necrosis of the tissue. Therefore, enniatin production by the enniatin-synthesizing strains may affect their pathogenicity. The enniatin synthetase gene (esyn1) of Fusarium scirpi ETH 1536 was used as a probe to determine if similar sequences were present in the strains examined. In Southern blot analyses, DNA sequences hybridizing with the esyn1 probe were present in all but two of the strains examined. In some cases, enniatin-nonproducing strains had the same hybridization pattern as enniatin producers.