Sediment–water partitioning behavior and bioavailability of five polycyclic aromatic hydrocarbons (PAHs; phenanthrene, pyrene, chrysene, benzo[k]fluoranthene, and benzo[a]pyrene) were measured in field-contaminated sediment collected from moderately polluted regions of the Anacostia River (Washington, DC, USA). Much of the sediment PAH burden was resistant to desorption: Effective partition coefficients were 2- to 10-fold greater than expected from literature values, and more than 80% of PAHs remained sorbed after treatment of the sediment with a nonionic polymeric adsorbent (Amberlite XAD-2) for 20 h. Bioaccumulation, elimination, and assimilation of each PAH in the deposit-feeding tubificid oligochaete Ilyodrilus templetoni were measured and compared with the equivalent measurements from laboratory-inoculated sediment. Ilyodrilus templetoni effectively accessed the desorption-resistant fraction of these organic contaminants, as exhibited by high single-gut passage assimilation efficiencies (ASEs) of the five PAHs (60% < ASE < 90%). However, steady-state accumulations of PAHs by I. templetoni were very low and consistent with low pore-water concentrations. The present results suggest that steady-state accumulation of PAHs is controlled by pore-water concentrations and is not necessarily related to route of uptake or assimilation efficiencies.