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In this article we present the new open-air Middle Pleistocene locality Marathousa 2, which was discovered during a double intensive and targeted field survey in the lignite mines of the Megalopolis basin (Greece). The locality is situated just below the Lignite Seam III of the Marathousa Member (Choremi Formation), and its similar stratigraphic position with the chronologically well-constrained locality Marathousa 1 (dated between 500 and 400 ka and correlated to the Marine Isotope Stage 12) indicates a comparable age for Marathousa 2. The locality most notably yielded dental and postcranial remains of Hippopotamus, with those found at the locality’s Area A most likely belonging to a single individual. The dimensions of the dental elements support an attribution to H. antiquus, a large-sized taxon, almost twice as large as the recent common H. amphibius, and with strong aquatic habits. Cut marks identified on a thoracic vertebra indicate hominin butchering of the carcass. Preliminary paleoenvironmental remarks based on the mammal and mollusk fauna, indicate that hominin activities took place at the margins of or near a perhaps cold freshwater water body, but with temperatures that allowed its persistence throughout the year, and perhaps under drier conditions compared to preceding and succeeding periods in the basin. In addition to the exploitation of elephants in Marathousa 1, Marathousa 2 provides further evidence of megafauna exploitation during the Middle Pleistocene in the Megalopolis Basin and one of the few examples of hippopotamus carcass processing during the Lower Paleolithic in Europe. Therefore, it advances our knowledge of the food acquisition strategies and subsistence behavior of Pleistocene Homo, and highlights the need for further research in the basin, where megafauna is well recorded.