IL-12 is a heterodimeric cytokine composed of an α-chain (p35) and a β-chain (p40). Primarily produced by APCs, IL-12 induces IFN-γ production in T, B and NK cells. IL-12 drives Th1-cell differentiation and IFN-γ secretion to promote cell-mediated immunity, which is essential in the defence against intracellular pathogens. The importance of IL-12 in Th1 responses is echoed by its targeted suppression by intracellular pathogens evading cell-mediated immunity. IL-12 subunits have been identified recently in fish, although reported bioactivities are limited to higher vertebrates. Here, we report the cloning of a p35 gene and two divergent p40 genes (p40b and p40c), capable of producing two functional IL-12 isoforms (p35/p40b and p35/p40c) in rainbow trout. Trout IL-12 isoforms possess distinct bioactivities with respect to the induction of IFN-γ, IL-10 and p40c expression. Trout IL-12 isoforms were differentially expressed and modulated in vivo, exhibiting specific gene expression profiles in bacterial, viral and parasitic infection models, and in vitro in stimulated macrophage and leucocyte cultures. These data imply that alternative or additional pathogen-specific Th-like cell populations may exist in fish. This study will facilitate a broader understanding of the evolutionary processes driving host-pathogen interactions and Th1-like immune responses in lower vertebrates. This article is protected by copyright. All rights reserved.