The highly fossiliferous sediments of the Tolomosa Formation from Tarija, southern Bolivia, represent one of the most important localities in South America that documents the Great American Biotic Interchange. Over the past several decades, chronostratigraphic studies have indicated a middle Pleistocene age for the Tolomosa Formation from ~ 1.1 to 0.7 Ma. This interval correlates to the Ensenadan South American Land Mammal Age as it is characterized from classic localities in Argentina. Recently, however, a new interpretation based on AMS 14C ages indicates that the fossiliferous sediments from Tarija are latest Pleistocene, i.e., < 44 ka, and thus of Lujanian age. Here we report a new age of 0.76 ± 0.03 Ma (2σ) based on 11 U–Th/Pb and U–Th/He individual determinations from the Tolomosa Formation. This is indistinguishable from the age published from the same ash in 1983, and was originally used to calibrate the magnetostratigraphic section at Tarija. The new age confirms that the age of the Tolomosa Formation is middle Pleistocene, and not latest Pleistocene. The age of the Tarija Fauna has significant implications with regard to the stage of evolution biochronology for Pleistocene fossil mammals in South America, and in particular, the classic and important reference sections in Argentina.