The present study investigated the bioaccumulation of the synthetic hormone 17 a-ethinylestradiol(EE2) in the benthic invertebrates Chironomus tentans and Hyalellaazteca, in water-only and spiked sediment assays. Water and sediment residue analysis was performed by LC/MS–MS, while biota extracts were analyzed using both LC/MS–MS and a recombinant yeast estrogen receptor assay. At the lowest exposure concentration, C. tentans accumulated less EE2 than H. azteca in the water-only assays (p ¼ 0.0004), but due to different slopes,this difference subsided with increasing concentrations; at the exposure concentration of 1mg/L, C. tentans had a greater body burden than H. azteca (p ¼ 0.02). In spiked sediments, C. tentans had the greatest EE2 accumulation(1.270.14vs.0.570.05 mg/gdw, n ¼ 4). Measurements in H. azteca indicated a negligible contribution from the sediments to the uptake of EE2 in this species. These differences were likely due to differences in the behavior and life history of the two species(epibenthicvs.endobenthic). Water-only bioaccumulation factors(BAFs)calculated at the lowest exposure concentration were significantly smaller in C. tentans than in H. azteca (31vs.142, respectively; po0.0001).In contrast,the sediment bioaccumulation factor(BSAF)of C. tentans was larger than that of H. azteca (0.8 vs.0.3; po0.0001). Extracts of the exposed animals caused a response in a recombinant yeast estrogen receptor assay,thus confirming the estrogenic activity of the samples,presumably from EE2 and its estrogenic metabolites. The results of the present study suggest that consumption of invertebrate food items could provide an additional source of exposure to estrogenic substances invertebrate predators.