Tumor necrosis factor alpha (TNFα) is a cytokine involved in a broad spectrum of cellular and organismal responses. Its main function, as a potent pro-inflammatory mediator, has been demonstrated in numerous teleost species and there are many reports on the modulation of TNFα gene expression under pathological conditions. Nevertheless, there is still scarce knowledge about the tissue distribution and type of cells that express this cytokine in fish species, which would help to further investigate its biological activities. These studies are hampered by the lack of molecular markers for teleost that hinder the development of morphological techniques, like immunohistochemistry. The aim of this work was to develop an immunohistochemical technique for the detection of TNFα in paraffin-embedded organs from healthy turbot (Scophthalmus maximus), an economically-important marine fish species. A commercial anti-human TNFα antibody, whose specificity was confirmed by western blot analysis, was used. Immunoreactive cells were observed in higher numbers in the lymphohematopoietic organs, kidney, spleen and thymus, although TNFα-positive cells were also present in the digestive tract, liver, heart, gills and skin. Similarly to non-fish species, monocytes/macrophages appeared to be the main producers of this cytokine; nevertheless, the presence of immunoreactive rodlet cells in different tissues was also reported. The nature and distribution of the labeled cells appeared to be related with a strategic localization for defense response to antigenic challenge. The relative abundance of TNFα-positive cells in the lymphohematopoietic organs also suggests that this cytokine may have a broader role in the normal physiology of those organs. The immunohistochemical technique allowed the in-situ characterization of TNFα expression, representing a valid tool to investigate the immune response of turbot.