Laboratory methods for measuring bioaccumulation of organic contaminants from sediment into aquatic organisms continue to improve, but some aspects are still in need of standardization. From a review of published methods, we noted that the loading density of organisms was determined inconsistently and was primarily based on either sediment volume or total organic carbon (TOC). The rationale mainly expressed for standardizing to TOC was to minimize the depletion of sediment contaminants. However, even when density was standardized to TOC, the relative amount of TOC provided (i.e., ratio of TOC to organism dry weight [dw]) was highly variable. In this study, we examined the effect of organism density (standardized to sediment TOC or volume) on bioaccumulation in three freshwater organisms. The oligochaete Lumbriculus variegatus, mayfly nymph Hexagenia spp., and fathead minnow Pimephales promelas were exposed for 28 days to two field-contaminated sediments that varied in concentration of PCBs and TOC. Densities tested were 50:1 and 27:1 ratios of TOC to organism dw and 140 ml sediment/g wet weight (ww) biomass, yielding low to high organism densities. Bioaccumulation in Hexagenia spp. was significantly higher at the lowest organism density compared with the highest organism density when exposed to site 2 sediment (1.1% TOC) but only with tissue concentrations expressed on a ww basis. Otherwise, there was no significant effect of density on bioaccumulation in organisms exposed to sediments from site 1 (12% TOC) or site 2. Survival of Hexagenia spp. was adversely affected at the highest organism density when the relative amount of TOC was low. The results of this study support the recommendation of standardizing organism density relative to a particular amount of TOC for invertebrate species. A 27:1 ratio of TOC:organism dw was selected as a standard organism density for a new bioaccumulation method because survival, growth, and bioaccumulation were not impacted relative to a 50:1 ratio, and less sediment was required. This density is recommended as an appropriate ratio for sediment bioaccumulation assessments in general.