To characterize the partitioning of metals in a stream ecosystem, concentrations of trace metals including As, Cd, Cu, Pb, and Zn were measured in water, colloids, sediment, biofilm (also referred to as aufwuchs), macroinvertebrates, and fish collected from the Boulder River watershed, Montana. Median concentrations of Cd, Cu, and Zn in water throughout the watershed exceeded the U.S. EPA acute and chronic criteria for protection of aquatic life. Concentrations of As, Cd, Cu, Pb, and Zn in sediment were sufficient in the tributaries to cause invertebrate toxicity. The concentrations of As, Cu, Cd, Pb, and Zn in invertebrates from lower Cataract Creek (63, 339, 59, 34, and 2,410 μg/g dry wt, respectively) were greater than the concentrations in invertebrates from the Clark Fork River watershed, Montana (19, 174, 2.3, 15, and 648 μg/g, respectively), that were associated with reduced survival, growth, and health of cutthroat trout fed diets composed of those invertebrates. Colloids and biofilm seem to play a critical role in the pathway of metals into the food chain and concentrations of As, Cu, Pb, and Zn in these two components are significantly correlated. We suggest that transfer of metals associated with Fe colloids to biological components of biofilm is an important pathway where metals associated with abiotic components are first available to biotic components. The significant correlations suggest that Cd, Cu, and Zn may move independently to biota (biofilm, invertebrates, or fish tissues) from water and sediment. The possibility exists that Cd, Cu, and Zn concentrations increase in fish tissues as a result of direct contact with water and sediment and indirect exposure through the food chain. However, uptake through the food chain to fish may be more important for As. Although As concentrations in colloids and biofilm were significantly correlated with As water concentrations, As concentrations in fish tissues were not correlated with water. The pathway for Pb into biological components seems to begin with sediment because concentrations of Pb in water were not significantly correlated with any other component and because concentrations of Pb in the water were often below detection limits.