In situ toxicity and bioaccumulation tests with Ceriodaphnia dubia (48 h), Chironomus tentans (96 h), Hyalella azteca (96 h), and Lumbriculus variegatus (96 h) were conducted at three stations on a river that was contaminated primarily with chlorobenzenes (CBs), and results were compared to a nearby reference site. Exposures were characterized by using minipiezometers for contaminant profiling and determination of hydraulic heads and vertical flow direction within the sediments and measuring contaminants in sediment, surface water, and exposure chamber water samples. Localized zones of upwelling and downwelling existed in the exposure areas at contaminated sites 5 and 18, while site 23 was downwelling at all measurement positions. Porewater samples from minipiezometers contained CBs at the three contaminated sites that were highest at site 23. However, sediment and water samples from exposure chambers at site 23 contained the lowest levels of CBs among the contaminated sites. The CBs were not detected at the reference site, but other organic contaminants and metals were detected at all sites, with the highest concentrations occurring at sites 5 and 18. In water column exposures, no significant (p > 0.05) differences were observed in species survival between the contaminated sites and the reference. Mean percentage survival of H. azteca, C. dubia, and C. tentans exposed to surficial sediments (SS) at sites 5 and 18 was significantly (p < 0.05) reduced compared to the reference, whereas only C. tentans survival was significantly reduced at site 23. Body residues of total CB congeners in L. variegatus exposed to SS were highest at site 18 (618 μmol/kg lipid) and lowest at site 23 (21 μmol/kg lipid). The data suggest that downwelling reduced the bioavailability of CBs in surficial sediments, most likely by mobilizing the freely dissolved and colloid-bound fractions to deeper sediments. Overall, downwelling conditions reduced the in situ exposure of organisms in surficial sediments and hence the toxicity and bioaccumulation of CBs. Hydrologic and chemistry data from nested minipiezometers improved the interpretation of exposure-effects relationships.