A cDNA library of mRNA from flounder leukocytes stimulated with bacterial lipopolysaccharide (LPS) and hemagglutinin was constructed to clone cytokine genes of this fish. Initial screening of this library with human cytokine gene probes was not productive and clones with inserts of over 400 nucleotides (nt) were randomly sequenced, and a homologue of the vertebrate interleukin-8 (IL-8) gene was isolated. The flounder IL-8 cDNA encompassed 884 nt, including a coding region of 330 nt. Four cysteines characteristic of CXC chemokines were identified at conserved locations in the putative protein. The deduced amino acid sequence showed 36 and 35% sequence identity with counterpart genes in monkey and human, respectively, and 52% sequence similarity with these genes. However, the putative flounder IL-8 amino acid sequence showed 25% identity and 52% similarity to that of lamprey, the only other piscine IL-8 gene that has been cloned. Flounder IL-8 transcripts were detected in the head-kidney and spleen of LPS-injected flounder and leukocytes stimulated with LPS. It was not detected in the muscle or liver of LPS-injected flounder, tissues taken from non-stimulated flounder and non-stimulated leukocytes.