The discrepancy between the ecological significance of amphipods in the Antarctic and our poor knowledge of their ecofunctional role calls for a more detailed investigation of their trophic status in this ecosystem. A total of 12 amphipod species from suspension feeder to scavenger have been considered in this study. Our objective was to investigate whether the combination of fatty-acid and stable-isotope signatures into a 2-dimensional trophic biomarker assay would increase accuracy in the identification of Antarctic benthic amphipod trophic position. Amphipod isotopic averages ranged from -29.3‰ (δ13C) and 4.1‰ (δ15N) for the suspension feeder Ampelisca richardsoni to -21.7‰ (δ13C) and 11.9‰ (δ15N) for the high predator Iphimediella sp. Cluster analysis of the fatty-acid composition separated the amphipod species into 4 trophic groups: suspension feeders, macro-herbivores, omnivores and scavengers. The suspension feeder was isolated due to an important proportion of 18:4(n-3), a fatty-acid biomarker of phytoplankton. Macro-herbivores were found to rely heavily on macroalgal carbon, containing a high percentage of arachidonic acid (20:4(n-6)). Scavenger amphipods revealed a unique fatty-acid composition dominated by 1 single fatty acid, 18:1(n-9), probably the result of a very intensive de novo biosynthesis to cope with starvation periods. Our data emphasise the need to combine different types of information to be able to draw the right conclusions regarding trophic ecology. Indeed, in some cases, the exclusive use of 1 type of tracing method, fatty acids or stable isotopes, would have resulted in misleading/false conclusions in the trophic classification of amphipods. Therefore, a 2-dimensional biomarker assay is a useful tool to elucidate the trophic positions of benthic amphipods. © Inter-Research 2005.