We characterized the biomolecules involved in handling cytosolic metals in larvae of the phantom midge (Chaoborus) collected from five mining-impacted lakes by determining the distribution of Ag, Cd, Cu, Ni, Tl, and Zn among pools of various molecular weights (HMW: high molecular weight, \textgreater670-40 kDa; MMW: medium molecular weight, 40-\textless1.3 kDa; LMW: low molecular weight, \textless1.3 kDa). Appreciable concentrations of nonessential metals were found in the potentially metal-sensitive HMW (Ag and Ni) and LMW (Tl) pools, whereas the MMW pool, which includes metallothioneins (MTs) and metallothionein-like proteins and peptides (MTLPs), appears to be involved in Ag and Cd detoxification. Higher-resolution fractionation of the heat-stable protein (HSP) fraction revealed further differences in the partitioning of nonessential metals (i.e., Ag = Cd ≠ Ni ≠ Tl). These results provide unprecedented details about the metal-handling strategies employed by a metal-tolerant, freshwater animal in a field situation. © 2016 American Chemical Society.