This study was designed to identify toxic ambient areas in the Chesapeake Bay watershed by using the following three estuarine and two freshwater 8-d water column toxicity tests: sheeps-head minnow, Cyprinodon variegatus, larval survival and growth test; grass shrimp, Palaemonetes pugio, larval survival and growth test; copepod, Eurytemora affinis, life cycle test; fathead minnow, Pimephales promelas, larval survival and growth test; and Ceriodaphnia dubia, survival and reproduction test. Ambient water was collected from the following locations and transported back to our laboratory for testing: Elizabeth River (VA), Patapsco River (MD), Wye River (MD), and Potomac River (three freshwater and two saltwater stations). The Potomac River stations were located at Indian Head (MD), Freestone Point (VA), Possum Point (VA), Morgantown (MD), and Dahlgren (VA). A suite of inorganic and organic contaminants and water quality conditions were evaluated in ambient water during the tests. Results from the water column tests demonstrated no significant ranking of sensitivity among the three saltwater tests, but rather supported the need for multispecies tests because different species displayed varying sensitivity to different types of contaminants. Biological effects were reported from at least one test species tested in ambient water from Elizabeth River, Patapsco River, Indian Head, Morgantown, and Dahlgren. These results demonstrate that selected ambient areas of the Chesapeake Bay watershed are toxic, based on various biological indicators.