High-salinity groundwater more than 1,000 metres deep in the Atlantic coastal plain of the USA has been documented in several locations(1,2), most recentlywithin the 35-million-year-old Chesapeake Bay impact crater(3-5). Suggestions for the origin of increased salinity in the crater have included evaporite dissolution(6), osmosis(6) and evaporation from heating(7) associated with the bolide impact. Here we present chemical, isotopic and physical evidence that together indicate that groundwater in the Chesapeake crater is remnant Early Cretaceous North Atlantic (ECNA) sea water. We find that the sea water is probably 100-145 million years old and that it has an average salinity of about 70 per mil, which is twice that of modern sea water and consistent with the nearly closed ECNA basin(8). Previous evidence for temperature and salinity levels of ancient oceans have been estimated indirectly from geochemical, isotopic and palaeontological analyses of solid materials in deep sediment cores. In contrast, our study identifies ancient sea water in situ and provides a direct estimate of its age and salinity. Moreover, we suggest that it is likely that remnants of ECNA sea water persist in deep sediments at many locations along the Atlantic margin.