A CC chemokine gene (JFCCL3) was cloned and sequenced from Japanese flounder, Paralichthys olivaceus. The JFCCL3 cDNA contains an open reading frame of 288 nucleotides encoding 95 amino acid residues. The predicted amino acid sequence of JFCCL3 showed the conserved cysteine of the beta chemokine plus two additional cysteines. The genomic sequence consists of two isoforms: JFCCL3.1 and JFCCL3.2 with sizes of 1.8 and 1.2kb, respectively. Both isoforms contain three introns and four exons. RT-PCR showed that JFCCL3 is constitutively expressed in most tissues including lymphoid organs. Using quantitative real-time RT-PCR, the highest expression of JFCCL3 transcripts was observed in PBLs at 3h post-stimulation with Con A/PMA and at 1h post-stimulation with LPS. A phylogenetic analysis showed that JFCCL3 is more closely related to fractalkines than to other mammalian beta chemokines. A chemotaxis assay showed that recombinant JFCCL3 protein has bioactivity for Japanese flounder leukocyte attraction at concentrations from 0.01 to 10 microg/ml.