: An EST database of more than 900 sequences has been constructed from complementary DNAs from six different tissues (stomach, intestine, pyloric cecum, liver, spleen, and ovary) of the winter flounder Pleuronectes americanus. Template preparation and automated sequencing were optimized to generate high-quality information in an economic fashion. Using computer scripts developed in our laboratory, the sequences were automatically compared with sequences in the databases via a Web-browser interface, and significant returns were recorded and organized on user-friendly HTML pages. Half (453) of the ESTs had significant matches to database sequences of known function, 33 matched ESTs from other organisms, 34 matched ribosomal RNAs, and 24 matched hypothetical open reading frames of unknown function. Forty-one percent (374) of the ESTs had no matches to sequences in the database and presumably represent previously unidentified cDNAs. Several sequences are the first isolated from teleost fish, and should be of interest for gene mapping and studies of developmental biology.