This study was conducted to support a Natural Resource Damage Assessment and Restoration project associated with the Ashtabula River in Ohio. The objective of the study was to evaluate the chemistry and toxicity of 50 sediment samples obtained from five cores collected from the Ashtabula River (10 samples/core, with each 10-cm-diameter core collected to a total depth of about 150 cm). Effects of chemicals of potential concern (COPCs) measured in the sediment samples were evaluated by measuring whole-sediment chemistry and whole-sediment toxicity in the sediment samples (including polycyclic aromatic hydrocarbons [PAHs], polychlorinated biphenyls [PCBs], organochlorine pesticides, and metals). Effects on the amphipod Hyalella azteca at the end of a 28-day sediment toxicity test were determined by comparing survival or length of amphipods in individual sediment samples in the cores to the range of responses of amphipods exposed to selected reference sediments that were also collected from the cores. Mean survival or length of amphipods was below the lower limit of the reference envelope in 56% of the sediment samples. Concentrations of total PCBs alone in some samples or concentrations of total PAHs alone in other samples were likely high enough to have caused the reduced survival or length of amphipods (i.e., concentrations of PAHs or PCBs exceeded mechanistically based and empirically based sediment quality guidelines). While elevated concentrations of ammonia in pore water may have contributed to the reduced length of amphipods, it is unlikely that the reduced length was caused solely by elevated ammonia (i.e., concentrations of ammonia were not significantly correlated with the concentrations of PCBs or PAHs and concentrations of ammonia were elevated both in the reference sediments and in the test sediments). Results of this study show that PAHs, PCBs, and ammonia are the primary COPCs that are likely causing or substantially contributing to the toxicity to sediment-dwelling organisms.