We generated 196 expressed sequence tags (EST) from a Japanese eel Anguilla japonica spleen cDNA library. Of these, 90 EST (46%) showed no homology to known genes, while 106 EST (54%) showed homology to known genes and transcripts. Of the 106 homologous EST, 13 were homologous to 12 different immune-related genes, 46 were homologous to 33 different ribosomal proteins, three were homologous to the 28S rRNA gene, 10 were homologous to eight different mitochondrial genes and the remaining 34 encoded 28 different identified genes. Six identified EST are similar to previously reported EST of Japanese flounder Paralichthys olivaceus spleen cDNA. These analyses demonstrate the utility of EST and sequence-tagged clones in comparative studies of gene expression in the fish spleen.