We evaluated exposure of aquatic biota to lead (Pb), zinc (Zn), and cadmium (Cd) in streams draining a Pb-mining district in southeast Missouri. Samples of plant biomass (detritus, periphyton, and filamentous algae), invertebrates (snails, crayfish, and riffle benthos), and two taxa of fish were collected from seven sites closest to mining areas (mining sites), four sites further downstream from mining (downstream sites), and eight reference sites in fall 2001. Samples of plant biomass from mining sites had highest metal concentrations, with means 10- to 60-times greater than those for reference sites. Mean metal concentrations in over 90% of samples of plant biomass from mining sites were significantly greater than those from reference sites. Mean concentrations of Pb, Zn, and Cd in most invertebrate samples from mining sites, and mean Pb concentrations in most fish samples from mining sites, were also significantly greater than those from reference sites. Concentrations of all three metals were lower in samples from downstream sites, but several samples of plant biomass from downstream sites had metal concentrations significantly greater than those from reference sites. Analysis of supplemental samples collected in the fall of 2002, a year of above-average stream discharge, had lower Pb concentrations and higher Cd concentrations than samples collected in 2001, near the end of a multi-year drought. Concentrations of Pb measured in fish and invertebrates collected from mining sites during 2001 and 2002 were similar to those measured at nearby sites in the 1970s, during the early years of mining in the Viburnum Trend. Results of this study demonstrate that long-term Pb mining activity in southeast Missouri has resulted in significantly elevated concentrations of Pb, Cd, and Zn in biota of receiving streams, compared to biota of similar streams without direct influence of mining. Our results also demonstrate that metal exposure in the study area differed significantly among sample types, habitats, and years, and that these factors should be carefully considered in the design of biomonitoring studies.