Granulocyte-colony stimulating factor (G-CSF) is a cytokine that stimulates the proliferation and differentiation of hematopoietic progenitor cells committed to the neutrophil/granulocyte lineage. Here, we report the two distinct granulocyte colony stimulating factor homologues from black rockfish Sebastes schlegelii. The G-CSF homologue cDNAs were isolated from the black rockfish LPS or Con A/PMA stimulated leukocyte cDNA libraries. The cDNA for the Black rockfish G-CSF-1 homologue predicts a peptide of 202 amino acids that is the closest to the Bastard halibut (Paralichthys olivaceus) G-CSF, whereas the cDNA of the Black rockfish G-CSF-2 homologue predicts a peptide of 212 amino acids that is the closest to the Fugu (Takifugu rubripes). In a healthy fish, the mRNAs of both G-CSF homologues were predominantly expressed in leukocytes, spleen, and gill. Expression of the black rockfish G-CSF-1 homologue was induced in peripheral blood leukocytes (PBLs) after stimulation with LPS, Con A/PMA, or Poly I:C, and G-CSF-2 homologue was strongly induced in PBLs after stimulation with Con A/PMA for 24 h only.