Subtractive hybridization was used to study river lamprey (Lampetra fluviatilis) leukocyte-specific cDNA. A clone representing the most abundant component (12%) of the leukocyte library subtracted with liver cDNA was isolated and characterized. The cDNA encodes a presumably secreted polypeptide of 101 residues. The 3' untranslated region of the cDNA contains motifs characteristic of the transiently expressing genes. Comparison of the deduced amino acid sequence with known protein sequences revealed its homology to the members of the chemokine superfamily. Designated as LFCA-1, the lamprey protein contains four conserved cysteines, of which the first two are separated by a residue, and a number of other CXC family characteristic residues. LFCA-1 has the highest similarity to the chicken EMF-1 (40%) and to the mammalian IL-8 (32-33%). However, it lacks the ELR motif essential for the function of the mammalian IL-8-related chemokines. Based on the phylogenetic analysis of the LFCA-1 relationship to the higher vertebrate chemokines, it is concluded that the evolutionary origin of the chemokine superfamily is ancient, and that the divergence of the CXC and CC families most likely occurred at the time or before the first vertebrates emerged.