In the current study, we have determined the immune effects of the intramuscular injection of eukaryotic expression plasmids coding for rainbow trout (Oncorhynchus mykiss) CK5B, CK6 or CK7A CC chemokines (pCK5B, pCK6 and pCK7A) as a first step towards the establishment of their biological role. We have studied the levels of expression of several immune genes in the spleen and head kidney by real-time PCR in comparison to the levels observed in animals injected with the empty plasmid. Concerning the levels of expression of these CC chemokines and the CXC chemokine, interleukin 8 (IL-8), each plasmid induced up-regulation on expression levels of its coded chemokine in the head kidney and spleen, but also affected the expression of other chemokines. Both pCK6 and pCK7A induced the expression of the other two CC chemokines, while pCK5B induced CK7A but not CK6. Both pCK5B and pCK7A induced IL-8 as well. pCK6 was the only plasmid that induced IL-1beta in the head kidney, whereas in the spleen, this occurred only with pCK5B. Different effects on the head kidney and spleen were also visible for tumour necrosis factor alpha (TNF-alpha), since the three plasmids induced this cytokine in the head kidney, but only pCK5B and pCK6 in the spleen. Concerning the effects on type I interferon (IFN), again pCK6 induced the strongest enhancement in the head kidney, while in the spleen it was pCK5B. However, the levels of expression of the Mx gene, know to be induced by type I IFN correlated with the CK6-induced IFN levels in the head kidney, but not with the CK5B-induced IFN in head kidney or spleen, suggesting an inhibition of Mx mRNA levels independent of IFN due to CK5B. The clear effect of pCK6 on the levels of expression of IFN-gamma and its strong effects on type I IFN, in contrast with its recent adscription to the CCL17/22 group linked to Th2 responses, were verified by studying the in vitro effects of recombinant CK6 on head kidney leukocytes. Again in this case, recombinant CK6 strongly induced type I IFN, Mx and IFN-gamma to a lesser extent, revealing CK6 as a potent IFN inducer in contrast to its mammalian homologues. Finally, effects on major histocompatibility complex (MHC)-IIalpha, CD4 and CD8alpha expression demonstrate that the three chemokines are able to mobilize antigen-presenting cells, CD4(+) and CD8(+) lymphocytes.