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The Wilms’ tumour gene (WT1) has previously been described as an oncogene in several neoplasms of humans, including melanoma, and its expression increases cancer cell proliferation. Recent reports associate the expression of the PPARβ/δ gene (peroxisome proliferator-activated receptor beta/delta) with the downregulation of WT1 in human melanoma and murine melanoma cell lines. The aim of this work was to analyse the expression of WT1 and its association with PPARβ/δ in samples of healthy and melanoma-affected skin of horses by immunohistochemistry. WT1 protein expression was detected in healthy skin, mainly in the epidermis, hair follicle, sebaceous gland and sweat gland, while no expression was observed in equine melanoma tissues. Moreover, it was observed that PPARβ/δ has a basal expression in healthy skin and that it is overexpressed in melanoma. These results were confirmed by a densitometric analysis, where a significant increase of the WT1-positive area was observed in healthy skin (128.66 ± 19.84 pixels 106) compared with that observed in melanoma (1.94 ± 0.04 pixels 106). On the other hand, a positive area with an expression of PPARβ/δ in healthy skin (214.94 ± 11.85 pixels 106) was significantly decreased compared to melanoma (624.86 ± 181.93 pixels 106). These data suggest that there could be a regulation between WT1 and PPARβ/δ in this disease in horses.