Interleukin-10 (IL-10) is a multifaceted cytokine that is produced by and effects a variety of cell populations, including macrophages, T, B and NK cells. The gene encoding for IL-10 has been isolated in mammals, birds, amphibians and recently in fish, with only single copy identified in each species. We report here a second IL-10 gene (tIL-10b) in rainbow trout that showed 92% identity in the coding region but only 50% identity in the 5'- and 3'-UTR to the known trout IL-10 paralogue, which we have now called tIL-10a. There is a short upstream open reading frame (uORF) within the 5'-untranslated region (UTR) of tIL-10a that may inhibit its translation, whilst in tIL-10b multiple mRNA instability motifs exist in the 3'-UTR, suggesting that the two IL-10 paralogues may have different mechanisms to regulate their expression post-transcriptionally. The expression of tIL-10a is generally higher than that of tIL-10b in most of the fourteen tissues examined and in the RTS-11, RTL and RTGill cell lines. However, the expression level of tIL-10b can exceed that of tIL-10a, as seen in vivo in the ovary of healthy fish and in the gills of Yersinia ruckeri challenged fish, and in vitro in head kidney (HK) leucocytes cultured for ≥ 8 h. The expression of the trout IL-10 paralogues can be up-regulated by LPS and polyIC in RTS-11 cells and by LPS, polyIC, PHA, PMA, calcium ionophore (CI) and IL-21 in head kidney leucocytes, as well as by Y. ruckeri infection, and can be modulated positively or negatively by IFN-γ. Synergistic effects on up-regulation of IL-10 expression were also seen between PHA and IL-21, as well as between PMA and CI. The expression kinetics of the IL-10 paralogues was also found to be different, suggesting that rainbow trout has evolved different pathways to regulate the expression of the two IL-10 paralogues at the transcriptional level.