Expressed sequence tags (ESTs) were obtained from Japanese flounder (Paralichthys olivaceus). We present the results of single-pass sequencing of 493 ESTs from 350 clones from liver cDNA and 57 ESTs from 41 clones from spleen cDNA. Sequences of the cDNA clones were compared with sequences in the GenBank database. Two hundred and two clones (51.7%) appeared to be completely unknown and are likely to represent newly described genes, whereas 189 clones (48.3%) were identified based on matches to sequences in the databases. Three of the unidentified sequences were isolated from both the liver and spleen cDNA libraries. However, there were no identical sequences between liver and spleen clones.