The benthic bioavailability of surface sediment-associated polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) was quantified and assessed in Lake Ontario. Bottom sediments were collected from three areas along the Lake Ontario southern shoreline near Olcott, Rochester, and Oswego, New York. The sediment samples were subjected to 28-day, PCDD/F bioaccumulation experiments using the aquatic oligochaete Lumbriculus variegatus. Empirical data including only detectable PCDD/F tissue residues were used to quantify the benthic bioavailability of 11 PCDD/F congeners through the calculation of biota-sediment accumulation factors (BSAFs). BSAFs calculated for at least two lake areas were combined as Lake Ontario BSAFs for those specific congeners. Variability of the BSAFs was estimated by propagated error (PE) terms. Mean Lake Ontario BSAFs (±PE) ranged from 0.04±0.02 for octachlorodibenzofuran (OCDF) to 2.42±1.32 for 1,2,3,6,7,8-hexachlorodibenzofuran (HxCDF). Tetra-chlorodibenzo-p-dioxin (TCDD) and 1,2,3,4,7,8-HxCDF, shown to be among the highest toxic equivalencies (TEQs) in lake sediments, yielded BSAFs of 0.51±0.18 and 0.22±0.12, respectively. Statistical comparison of the TCDD BSAFs showed no significant differences among the lake areas. The benthic bioavailability of TCDD appeared lower than the results of other investigators. While hydrophobicity and degree of chlorination play some role, other factors, such as steric properties, and composition of organic carbon and matter, may have had a greater influence on the benthic bioavailability of PCDD/F congeners. Comparisons of these benthic BSAFs to fish BSAFs suggest that the bioaccumulation of PCDD/F congeners by fish is more related to water solubility and degree of chlorination than to sediment concentration.