To partially simulate conditions in fish intestinal tracts, we leached six groups of metals-contaminated invertebrates at pH 2 and pH 7, and analyzed the concentrations of four metals (Cd, Cu, Pb, and Zn) and total protein in the leachates. Four of the groups of invertebrates were benthic macroinvertebrates collected from metals-contaminated rivers (the Clark Fork River in Montana and the Coeur d’Alene River in Idaho, USA); the other two groups of invertebrates (one of which was exposed to metals in the laboratory) were laboratory-reared brine shrimp (Artemia sp.). Additionally, we fractionated the pH 2 leachates using size-exclusion chromatography (SEC). Protein content was 1.3 to 1.4× higher in Artemia than in the benthic macroinvertebrates, and leachability of metals and protein differed considerably among several of the groups of invertebrates. In SEC fractions of the pH 2 leachates from both groups of Artemia, Cu and protein co-eluted; however, Cu and protein did not co-elute in SEC fractions of the leachates from any of the benthic macroinvertebrate groups. Although none of the other three metals co-eluted with protein in any of the pH 2 leachates, one or more of the metals co-eluted with lower-molecular-weight molecules in the leachates from all of the groups of invertebrates. These results suggest fundamental differences in metal-binding properties and protein leachability among some invertebrates. Thus, different invertebrates and different histories of metals exposure might lead to different availability of metals and protein to predators.