Technical Abstract: The maize pathogen Fusarium verticillioides produces the fumonisin mycotoxins, which are of significant concern for their animal toxicity. This ascomycete is also reported from banana, but such strains do not produce fumonisins and are not pathogenic on maize seedlings. Southern analysis of several banana strains failed to detect genes in the fumonisin biosynthetic gene (FUM) cluster but did detect genes flanking the cluster. To determine whether the FUM cluster was absent in banana strains, the genomic sequence of strain NRRL 25059 was determined for genes flanking each side of the cluster as well as the intervening DNA. Sequence data indicated the FUM cluster was deleted with putative break points located upstream of FUM1 and within FUM19. PCR confirmed absence of the cluster in all 18 banana strains examined. To determine if complementation was possible, strain NRRL 25059 was cotransformed with two overlapping cosmids that together contain the entire FUM cluster. Only transformants carrying both cosmids produced fumonisins B1, B2, and B3 to levels comparable to wild-type maize strains. Cotransformants were also pathogenic on maize seedlings. For the first time documentation is provided for both a deletion of the FUM gene cluster and its functional, molecular genetic complementation. These data also support the importance of fumonisins for development of maize seedling disease caused by F. verticillioides.