Estrogenic substances discharged from wastewater treatment plants have been detected in surface sediments of receiving waters, but little is known of their vertical migration through buried sediments and their potential to contaminate subsurface waters. The vertical profiles of estrogenic chemicals were investigated in sediment cores at an alluvial freshwater site (Ditchling) and a clay-rich estuarine site (Lewes), both of which are downstream of wastewater discharges into the River Ouse (Sussex, U.K.). Estrone (E1) was the predominant estrogen detected in surface and buried sediments at both sites and was detected in undisturbed clay sediments >120 years old. Profiles of E1 at Ditchling were characterized by a prominent subsurface peak of E1 at the alluvium/clay interface (-15 cm) at a concentration (28.8 ± 6.0 ng/g of dry wt) that was 9-fold higher than in the surface sediment. In contrast, a steady downcore decline in E1 concentrations was observed in the clay-rich Lewes core. This work provides the first in situ evidence of estrogen migration through river bed sediments and reveals that movement of estrogens through unconsolidated sediment can result in penetration to the underlying substrata and therefore the potential for groundwater contamination.