Fusarium head blight, caused by the fungus Fusarium graminearum, is a major disease on wheat (Triticum aestivum L.). Expressed sequence tags (ESTs) were used to identify genes expressed during the wheat-F. graminearum interaction. We generated 4,838 ESTs from a cDNA library prepared from spikes of the partially resistant cultivar Sumai 3 infected with F. graminearum. These ESTs were composed of 2,831 singlet (single-copy transcripts) and 715 contigs (multiple-copy transcripts) for a total of 3,546 non-redundant sequences. Four sets of nonredundant sequences were identified. One set contains numerous, common biotic and abiotic stress-related genes. Many of these stress-related genes were represented by multiple ESTs, indicating that they are abundantly expressed. A second set comprised 16 nonredundant sequences from F. graminearum that may be required for pathogenicity. A subset of these fungal genes encodes proteins associated with plant cell wall degradation. A third set of 326 nonredundant sequences had no DNA or amino acid sequence similarity to almost 1 million plant and over 7 million animal sequences in dbEST (as of 22 June 2001). Thus, these 326 nonredundant sequences have only been found in our F. graminearum-infected ‘Sumai 3’ cDNA library. A fourth set of 29 nonredundant sequences was found in our F. graminearum-infected wheat and another plant-pathogen interaction cDNA library. Some of these sequences encode proteins that may act in establishing various plant-fungal interactions.