Certain heavy metal actions such as Cd2+ and Pb2+ mimic Ca2+ effectively in stimulating calmodulin (CaM). We now show that these cations also activate skeletal muscle troponin C (TnC), a Ca2(+)-binding protein highly homologous to CaM. Like Ca2+, these cations allow TnC to alter its electrophoretic mobility on polyacrylamide gels, and to bind to phenyl-Sepharose. Moreover, they activate TnC to stimulate myofibrillar ATPase. When TnC was removed from the skeletal myofibrils by treatment with trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CDTA), the ATPase activity was no longer stimulated by the cations. However, after reconstitution of CDTA-treated skeletal myofibril with TnC, the response of ATPase to Ca2+, Cd2+ or Pb2+ was restored. These findings suggest that the activation of myofibrillar ATPase by Cd2+ and Pb2+ is mediated through TnC. The ability of the heavy metals to stimulate TnC-supported ATPase activity correlated quite well with the ability to increase the extent of the myofibrillar superprecipitation. The activation of TnC by Cd2+ or Pb2+ could constitute a possible molecular basis for their toxicity.