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Avian feathers have the potential to accumulate trace elements originating from contaminated food and polluted environments. In fact, in feathers, metals bind to keratin, a sulphur-containing protein for which several metals have a strong affinity. Here, the concentrations of 18 essential and non-essential elements were investigated in a Humboldt penguin (Spheniscus humboldti) colony housed at the Acquario di Cattolica (Italy). This species is listed as vulnerable in the Red List of the International Union for Conservation of Nature. According to the literature, there is usually a link between metal levels in the diet of birds and levels detected in their feathers. Thus, metals were also determined in the penguins’ food (capelin, Mallotus villosus). We hypothesize that the controlled conditions in which birds are kept in captivity, and the homogeneous diet that they follow could allow a better understanding of metal bioaccumulation (such as mercury) or bio-dilution (such as arsenic) in the marine food chain, indicated by penguins’ feathers. Moreover, comparisons with our previous investigations performed on an ex-situ African penguin (Spheniscus demersus) colony suggest that penguins living indoors have lower body burden of metals than those living outdoors. Indeed, environmental contaminants usually found in areas subjected to anthropogenic impact, where zoos and aquaria are often located, are not accumulated to levels of concern.