The Cape Fear shiner (Notropis mekistocholas) is a recently described cyprinid species endemic to the Cape Fear River Basin of North Carolina, USA. Only five populations of the fish remain; thus, it is listed as endangered by the U.S. Government. Determining habitat requirements of the Cape Fear shiner, including water quality and physical habitat, is critical to the survival and future restoration of the species. To assess water quality in the best remaining and in the historical habitats, we conducted a 28-d in situ bioassay with captively propagated Cape Fear shiners. Fish were deployed at 10 sites in three rivers, with three cages per site and 20 fish per cage. Water and sediment samples were collected and analyzed for selected metals and organic contaminants. Passive sampling devices also were deployed at each site and analyzed for organic contaminants at test termination. Fish survival, growth (as measured by an increase in total length), and contaminant accumulation were measured on completion of the bioassay. Survival of caged fish averaged 76% (range, 53–100%) and varied significantly among sites and rivers. Caged fish accumulated quantities of cadmium, mercury, polychlorinated biphenyls, and other persistent contaminants over the test duration and grew significantly at only four sites. No apparent relations were observed between exposure to or accumulation of a specific contaminant and reduced growth or survival of fish among all the sites. However, a generalized hazard assessment showed that certain sites exhibited trends in cumulative contaminant presence with reduced fish survival and growth, thereby enabling the identification of the existing riverine habitat most suitable for reintroduction or population augmentation of this endangered fish.