The frequency of Ty1- copia-type and Ty3- gypsy-type retrotransposons in the International Triticeae EST Consortium (ITEC) database (61,942 sequences: 82% wheat, 10% barley, 8% rye) and the DuPont EST database (86,628 wheat sequences) was estimated using BLASTN searches. These ESTs were obtained from 94 cDNA libraries from different tissues (leaves, roots, spikes, flowers and seeds) and different growing conditions, excluding subtracted and normalized cDNA libraries. Triticeae EST databases were screened using four different Ty1- -copia-type, 12 reverse transcriptase sequences, and three Ty3- gypsy-type Triticeae retrotransposon sequences. Using a selection threshold of BLASTN scores higher than 100 or E values smaller than e(-20), 0.145% of the ESTs were found to be significantly similar to at least one of the retrotransposons used in the search (0.064% Ty1- copia, 0.081% Ty3- gypsy). This percentage increased to 0.176% when the BLASTN threshold was changed to E<e(-10). The percentage of ESTs similar to retrotransposons was significantly higher ( P < 0.05) in cDNA libraries from leaf tissues than in cDNA libraries from roots, anthers, or spikes. In addition, the percentage of ESTs similar to retrotransposons in cDNA libraries from plants under stress conditions (0.25% at E<e(-20), and 0.30% at E<e(-10)) was three to four folds higher ( P < 0.0001) than in cDNA libraries from plants grown under normal conditions (0.07% at E<e(-20), and 0.09% at E<e(-10)). Identification of retrotransposons within the Triticeae EST databases provides an indirect estimation of the patterns of transcriptional activity of these repetitive elements and is important to improve the annotation of genomic sequences used to search these EST databases.