Lead (Pb) contamination of the environment remains a global problem. Previous studies have demonstrated that Pb deposited onto roadside sediments from the past use of leaded gasoline in vehicles may be mobilized into rivers and streams, thereby resulting in exposure to aquatic biota. The aims of this study were to conduct a 28-day laboratory toxicity test with Pb and adult Eastern Elliptio (Elliptio complanata; family Unionidae) mussels to determine uptake kinetics and to assess several potential non-lethal biomarkers of Pb exposure. Mussels were collected from a relatively uncontaminated reference site and exposed to a control and eight concentrations of Pb (as lead nitrate) ranging from 1 to 251 µg/L, as a static renewal test. There were five replicates per treatment with one mussel per replicate. The hemolymph of mussels from four of the replicates was repeatedly sampled (days 7, 14, 21, and 28) for analysis of Pb and ion (Na+, K+, Cl-, Ca2+) concentrations. The mussels in the fifth replicate per treatment were only sampled on day 28 and served as a comparison to the repeatedly sampled mussels. The accumulation of Pb in mussel tissue was also evaluated during the study. No mussels died during the test. We found that measured concentrations of Pb in mussel hemolymph suggested regulation of the heavy metal up to 66 μg/L by day 14, whereas concentrations in tissue proved to be strongly correlated (R2 = 0.98; p < 0.0001) throughout the 28-day exposure, displaying concentration dependent uptake. The concentration of Pb in mussel hemolymph, which can be sampled and measured non-lethally, is a suitable marker of recent Pb exposure in mussels. In contrast, none of the ion concentrations measured in the hemolymph from the repeatedly sampled mussels was significantly changed with increasing concentrations of Pb, whereas the mussels from the fifth replicate sampled only on day 28 showed altered calcium concentrations. The activity of δ-aminolevulinic acid dehydratase (ALAD), a demonstrated Pb-specific biomarker in vertebrates and some invertebrates, which was also evaluated as a potential endpoint in an initial evaluation for this study, proved to be an unsuitable biomarker in Elliptio complanata, with no detectable activity observed. This finding was in contrast to a second fre