To validate the standardization of a laboratory protocol for measuring bioaccumulation, laboratory-derived tissue residues and biota-sediment accumulation factors (BSAFs) were compared with historical field data from nine sites in Ontario, Canada. As a result of temporal discontinuity between the field and the laboratory studies, a priori considerations, such as changes in site conditions or concentrations of contaminants in sediment, were necessary to assess whether to compare absolute or relative measures of bioaccumulation. For the majority of sites, BSAFs for field-collected and laboratory-exposed fish were within a factor of 2. Biota-sediment accumulation factors for laboratory-exposed oligochaetes were typically greater than those for mussels caged in the field, by a factor of 2 to 9. Overall, the laboratory methods for all species generally overestimated the relative bioavailability of contaminants compared with field conditions by a factor of 1.1 to 9.2. Other than the great disparity observed for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) between field and laboratory studies, on average the laboratory-derived BSAFs with Pimephales promelas and Lumbriculus variegatus overestimated those from field-collected fish and field-exposed mussels by factors of 1.6 and 3.6, respectively. The laboratory method reflects a potentially worst-case exposure scenario and provides an appropriately conservative estimate of bioaccumulation. Laboratory-based estimates can be comparable to bioaccumulation data from the field but may be confounded by species-specific differences in routes of exposure and bioaccumulation of certain compounds or other environmental and biological factors that should be considered in these comparisons.