Trace metals are present in all aquatic systems, originating both from natural and anthropogenic sources. With concerns over the environmental impacts of metals, particularly in semi-enclosed aquatic systems such as the Port Phillip Bay, Victoria, Australia, it is important to be able to monitor the degree of pollutant exposure to local organisms. Little Penguins (Eudyptula minor) nesting at St Kilda, only 3 km from the centre of the 3.3 million people metropolis of Melbourne, are a potentially useful bioindicator species for toxicant exposure within Port Phillip Bay because they are resident and feed exclusively within the bay all year around. This study investigated metal and metalloid concentrations in Little Penguins at St Kilda and two other locations with different levels of anthropogenic impact: the penguin parade at Phillip Island and the remote off-shore Notch Island.