Abstract: In 2003, the U.S. Geological Survey (USGS) National Water-Quality Assessment (NAWQA) program and U.S. Environmental Protection Agency studied total mercury (THg) and methylmercury (MeHg) concentrations in periphyton at eight rivers in the United States in coordination with a larger USGS study on mercury cycling in rivers. Periphyton samples were collected using trace element clean techniques and NAWQA sampling protocols in spring and fall from targeted habitats (streambed surface-sediment, cobble, or woody snags) at each river site. A positive correlation was observed between concentrations of THg and MeHg in periphyton (r2 = 0.88, in log-log space). Mean MeHg and THg concentrations in surface-sediment periphyton were significantly higher (1,333 ng/m2 for MeHg and 53,980 ng/m2 for THg) than cobble (64 ng/m2 for MeHg and 1,192 ng/m2 for THg) or woody snag (71 ng/m2 for MeHg and 1,089 ng/m2 for THg) periphyton. Concentrations of THg in surface-sediment periphyton had a strong positive correlation with concentrations of THg in sediment (dry weight). The ratio of MeHg:THg in surface-sediment periphyton increased with the ratio of MeHg:THg in sediment. These data suggest periphyton may play a key role in mercury bioaccumulation in river ecosystems.