A large-scale analysis of expressed sequence tags (EST) was conducted in a unicellular green alga, Chlamydomonas reinhardtii. To complement the previously published EST sequences obtained from photoautotrophically grown cells and cells under CO(2) stress condition, a mixture of cells grown under various stress conditions including light, temperature, salt and limited nutrient was used as a material to construct the complementary DNA library. We have generated 12,842 5' end sequences from this library, making the collection 50,832 in its entirety. To speed up the data processing step, an internal semiautomatic operation pipeline was constructed. Using this system, quality check of the generated sequences, trimming, and database search for primary annotation was performed. Clustering of the EST sequences by BLAST followed by Phrap has produced 3748 consensus sequences and 7721 singletons. To make this high-quality set of assemblage information as well as clones available to the research community, a Web database with useful services was created at http://www.kazusa.or.jp/en/plant/chlamy/EST/.