Expressed sequence tag (EST) analysis is an efficient tool for gene discovery and for profiling gene expression. A cDNA library developed from messenger RNA of the Japanese flounder, Paralichthys olivaceus spleen was constructed by directional cloning, in order to isolate functional genes involved in immunity in fish. A total of 1010 ESTs from the library were sequenced and compared with sequences in the GenBank database. Of the 1010 ESTs, 618 ESTs (61%) were identified as being homologous with known genes from many organisms by BLAST searches, whereas 392 (39%) appeared to be unknown and are likely to represent newly described genes. Of the identified genes, 105 (17%) encoded proteins associated with cell/organism defence and homeostasis. Of these 105 genes, 21 were identified for the first time in Japanese flounder. These included macrophage inflammatory protein (MIP)-3 α, granulin-A, novel immune-type receptor (NITR), interferon regulatory factor (IRF)-10, presenilin-like protein-2, and antizyme inhibitor. A comparison of ESTs derived from spleen, kidney, liver and leukocytes suggested that expression of immune-related genes in different tissues, organs, or cells are different.KEY WORDS: cDNA sequence, expressed sequence tag, Japanese flounder, spleen.