Two different cDNA clones of Japanese flounder (types I-1 and I-2) with lengths of 1096 and 1572bp, respectively, were found to encode the same serine protease consisting of 244 identical amino acid residues with three putative N-glycosylation sites, an 18-amino acid signal peptide and a 2-amino acid activation peptide. The amino acid sequence of the Japanese flounder serine protease shares 39-44% identity to known hematopoietic serine proteases. Genomic analysis showed that two different clones were alternatively spliced from the same gene. A phylogenetic tree analysis showed that the Japanese flounder serine protease clustered with a hypothetical fugu protein and this cluster belonged to the neutrophil serine protease family cluster, which includes myeloblastin, N-elastase, and azurocidin. Expression of the Japanese flounder serine protease gene was observed to be up-regulated in head kidney cells after infection with Hirame rhabdovirus and LPS induction. In situ hybridization indicated that cells expressing Japanese flounder serine protease are different from CD8(+) and immunoglobulin(+) cells.