The unique Australian monotreme, the platypus (Ornithorhynchus anatinus) potentially exerts a strong top-down influence on riverine food webs in eastern Australia. However, despite considerable interest in the evolutionary history and physiology of the platypus, little is known of its trophic relationships. To address this lack of knowledge we used stable isotope analysis, in combination with the analysis of food items stored in cheek pouches, to determine its position in a typical riverine food web. This was the essential first step in the process of designing a larger study to investigate the relative importance of top-down and bottom-up effects in rivers where the platypus occurs. We found that platypuses were feeding on a wide range of benthic invertebrates, particularly insect larvae. The similarity of δ13C and δ15N values recorded for the platypus, a native fish (Galaxias sp.) and the exotic mosquitofish (Gambusia holbrooki) indicated dietary overlap and potential competition for the same resources. Although cheek pouch studies identify most of the major groups of prey organisms, the potential for contribution of the soft-bodied organisms such as larval dipterans, is suggested by stable isotope analysis, indicating that the use of both techniques will be important in future ecological investigations.