A group of expressed sequence tags (ESTs) from the wheat fungal pathogen Mycosphaerella graminicola utilizing ammonium as a nitrogen source has been analyzed. Single pass sequences of complementary DNAs from 986 clones were determined. Contig analysis and sequence comparisons allowed 704 unique ESTs (unigenes) to be identified, of which 148 appeared as multiple copies. Searches of the nrdb95 protein database at EMBL using the BLAST2x algorithm revealed 407 (57.8%) sequences that generated high to moderate high scoring pairs with proteins of known and unknown function. The rest of the sequences (297) showed either weak or no similarities to database entries. Among the unigenes with assigned function, 26.7% were involved in primary metabolism and 17.9% were associated with protein and RNA metabolism. Fewer clones were ascribed roles in signal transduction (4.9%), transport and secretion (6.1%), cell structure (3.1%), and cell division (3.6%). Approximately 18.1% of the identities found were to hypothetical or unknown proteins mainly from the yeasts Saccharomyces cerevisiae and Schizosaccaromyces pombe. Comparison of the 297 sequences with no clear function to other fungal ESTs in the public domain revealed 12 sequences that had high to moderate similarity to Neurospora crassa, Emericella (Aspergillus) nidulans, or Magnaporthe grisea sequences.