We assessed the exposure of fish from the Spring and Neosho Rivers in northeast Oklahoma, USA, to lead, zinc, and cadmium from historical mining in the Tri-States Mining District (TSMD). Fish (n = 74) representing six species were collected in October 2001 from six sites on the Spring and Neosho Rivers influenced to differing degrees by mining. Additional samples were obtained from the Big River, a heavily contaminated stream in eastern Missouri, USA, and from reference sites. Blood from each fish was analyzed for Pb, Zn, Cd, Fe, and hemoglobin (Hb). Blood also was analyzed for δ-aminolevulinic acid dehydratase (ALA-D) activity. The activity of ALA-D, an enzyme involved in heme synthesis, is inhibited by Pb. Concentrations of Fe and Hb were highly correlated (r = 0.89, p < 0.01) across all species and locations and typically were greater in common carp (Cyprinus carpio) than in other taxa. Concentrations of Pb, Zn, and Cd typically were greatest in fish from sites most heavily affected by mining and lowest in reference samples. The activity of ALA-D, but not concentrations of Hb or Fe, also differed significantly (p < 0.01) among sites and species. Enzyme activity was lowest in fish from mining-contaminated sites and greatest in reference fish, and was correlated negatively with Pb in most species. Statistically significant (p < 0.01) linear regression models that included negative terms for blood Pb explained as much as 68% of the total variation in ALA-D activity, but differences among taxa were highly evident. Positive correlations with Zn were documented in the combined data for channel catfish (Ictalurus punctatus) and flathead catfish (Pylodictis olivaris), as has been reported for other taxa, but not in bass (Micropterus spp.) or carp. In channel catfish, ALA-D activity appeared to be more sensitive to blood Pb than in the other species investigated (i.e., threshold concentrations for inhibition were lower). Such among-species differences are consistent with previous studies. Enzyme activity was inhibited by more than 50% relative to reference sites in channel catfish from several TSMD sites. Collectively, our results indicate that Pb is both bioavailable and active biochemically in the Spring-Neosho River system.