Comparison of new benthic foraminiferal δ18O and δ13C records from Ocean Drilling Program (ODP) Site 1263 (Walvis Ridge, southeast Atlantic, 2100 m paleodepth) and Deep Sea Drilling Project (DSDP) Site 366 (Sierra Leone Rise, eastern equatorial Atlantic, 2200–2800 m paleodepth) with published data from Atlantic and Southern Ocean sites provides the means to reconstruct the development of deep-water circulation in the southeastern Atlantic from the late-middle Eocene to the earliest Oligocene. Our comparison shows that in the late-middle Eocene (ca. 40 Ma), the South Atlantic was characterized by a homogeneous thermal structure. Thermal differentiation began ca. 38 Ma. By 37.6 Ma, Site 1263 was dominated by Southern Component Water; at the same time, warm saline deep water filled the deeper South Atlantic (recorded at southwest Atlantic ODP Site 699, paleodepth 3400 m, and southeast Atlantic ODP Site 1090, paleodepth 3200 m). The deep-water source to eastern equatorial Site 366 transitioned to Northern Component Water ca. 35.6–35 Ma. Progressive cooling at Site 1263 during the middle to late Eocene and deep-water thermal stratification in the South Atlantic may be attributed at least in part to the gradual deepening and strengthening of the proto–Antarctic Circumpolar Current from the late-middle Eocene to the earliest Oligocene, as the Drake and Tasman gateways opened. Our isotopic comparisons across depth and latitude provide evidence of the development of deep-water circulation similar to modern-day Atlantic Meridional Overturning Circulation.