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Multidisciplinary investigations at Pagliare di Sassa (L'Aquila, central Italy) suggest that the local succession accumulated from the late Early to the early Middle Pleistocene in a lacustrine environment. In the upper part, clastic sediments are part of an alluvial fan prograding into the lake, grading to a braided fluvial system. The pollen record confirms that a significant glacial phase occurred just before the onset of the Jaramillo inversion. These data, coupled with evidence from the nearby but earlier Madonna della Strada sequence, allow reconstruction of part of the environmental evolution of L'Aquila basin before the Jaramillo Subchron. The mammal species of Pagliare di Sassa include Stephanorhinus hundsheimensis, mostly of open environments, already present at Madonna della Strada. The faunal turnover characterizing the Early to Middle Pleistocene transition is indicated by the appearances of taxa typical of the Italian early to middle Galerian faunas, such as Praemegaceros verticornis, together with Megaloceros savini. The occurrence of Mimomys savini together with Microtus ex gr. Microtus hintoni-gregaloides suggests that this assemblage is earlier than the Isernia La Pineta fauna. A flint implement and a fragmentary herbivore femur with impact scars probably linked to human activity give evidence of the human peopling of intramontane basins of the Apennine chain since the early Middle Pleistocene.