Although many chemokine genes have been identified in rainbow trout (Oncorhynchus mykiss) as in other teleost species, almost no studies focused on their biological role have been conducted, despite the fact that no clear inferences as to their functions can be made based on their low similarity to mammalian counterparts. In the current work, we have studied the regulation of mRNA transcription and protein expression of CK12, a rainbow trout CC chemokine previously catalogued within the CCL19/21/25 phylogenetic group. Our studies revealed that CK12 is strongly expressed both at mRNA and protein level in mucosal tissues. Mature lymphocyte populations also express CK12 both at mRNA and protein levels. Concerning its biological activity, a significant chemotatic activity towards purified recombinant CK12 in unfractionated leukocyte populations was observed in the spleen, but not in head kidney or blood. Consequently, a binding assay revealed that the number of leukocytes capable of binding CK12 was much more elevated in spleen populations than in leukocyte populations from other organs. This binding capacity was only observed in small lymphocytes that should account for resident inactivated lymphocytes, in contrast to mature lymphocytes that were responsible for CK12 production. Around 36% of these small lymphocytes were IgM+ cells, of which 40% had a CK12 binding capacity. On the other hand, 10% of thymocytes were also capable of CK12 binding, suggesting that both T and B immature lymphocytes are recruited by CK12. This work constitutes the first description of a mucosal-associated chemokine in fish in which important aspects of its regulation and functionality are revealed.