The U.S. Geological Survey National Water-Quality Assessment Program conducted a multidisciplinary study to investigate the bioaccumulation of mercury from 2002 to 2004. Study areas were located in Oregon, Wisconsin, and Florida. Each study area included one urban site, and one or two nonurban sites that had the following attributes: high-percent wetland or low-percent wetland. Periphyton, macroinvertebrates, and forage fish were collected twice per year (during 2003 and 2004) to capture seasonality. Top predators, specifically largemouth bass (Micropterus salmoides), brown trout (Salmo trutta), and cutthroat trout (Oncorhynchus clarkii), were collected once per year (Oregon, Wisconsin, and Florida in 2003; Florida only in 2004). All biota were identified to the lowest possible taxonomic category and were analyzed for mercury and stable carbon and nitrogen isotopes. Periphyton and invertebrates were analyzed for total mercury and methylmercury; fish were analyzed for total mercury only. This report presents (1) methodology and data on mercury, methylmercury, stable isotopes, and (2) other ecologically relevant measurements in biological tissues of periphyton, invertebrates, forage fish, and predator fish.