Evidence of earliest human settlements observed in the alluvial formations of the Middle Loire Basin during systematic surveys organised since 1981. Many stepped fluvial terraces deposited during the successive interglacial–glacial Quaternary cycles have been identified in three tributary valleys of the Loire River: the Creuse, Cher and Loir Valleys. These alluvial remnants were systematically dated by the Electron Spin Resonance (ESR) method applied on optically bleached quartz, in order to construct a chronostratigraphical framework for the fluvial systems and the associated archaeological localities. Four localities containing Early Palaeolithic industries with Mode 1 technology, occupied during the Lower Pleistocene, and ten sites characterized by assemblages with handaxes, discovered in the Middle Pleistocene alluvial formations, are described in this paper. These two sets of localities are characterized by lithic industries with many differences in supply of raw materials and in technical behaviours. The results indicate that this area was reached by two separate phases of human settlement (one in relation to Mode 1, the other to Mode 2) with an interval around 400ka, at the end of the Lower Pleistocene, during a period characterized by major climatic degradations.