The divalent metal ion binding sites of skeletal myosin were investigated by electron paramagnetic resonance (EPR) spectroscopy using the paramagnetic (Mn(II) ion as a probe. Myosin possesses two high affinity sites (K less than 1 muM) for Mn(II), which are located on the 5,5'-dithiobis(2-nitrobenzoate) (DTNB) light chains. Mn(II) bound to the isolated DTNB light chain gives rise to an EPR spectrum similar to that of Mn(II) bound to myosin and this indicates that the metal binding site comprises ligands from the DTNB light chain alone. Myosin preparations in which the DTNB light chain content is reduced by treatment with 5,5'-dithiobis(2-nitrobenzoate) show a corresponding reduction in the stoichiometry of Mn(II) binding, but the stoichiometry is recovered on reassociation of the DTNB light chain. Chymotryptic digestion of myosin filaments in the presence of ethylenediaminetetraacetic acid yields subfragment 1, but digestion in the presence of divalent metal ions produces heavy meromyosin. Myosin with a depleted DTNB light chain content gives rise to subfragment 1 on proteolysis, even in the presence of divalent metal ions. It is proposed that saturation of the DTNB light chain site with divalent ions protects this subunit against proteolysis, which, in turn, inhibits the cleavage of the subfragment 1-subfragment 2 link. Either the DTNB light chain is located near the region of the link and sterically blocks chymotryptic attack, or it is bound to the subfragment 1 moiety and affects the conformation of the link region. When the product heavy meromyosin was examined by sodium dodecyl sulfate gel electrophoresis, an apparent anomaly arose in that there was no trace of the 19 000-dalton band corresponding to the DTNB light chain. This was resolved by following the time course of chymotryptic digestion of the myosin heavy chain, the DTNB light chain, and the divalent metal binding site. The 19 000-dalton DTNB light chain is rapidly degraded to a 17 000-dalton fragment which comigrates with the alkali 2 light chain. The divalent metal site remains intact, despite this degradation, and the 17 000 fragment continues to protect the subfragment 1-subfragment 2 link. In the absence of divalent metal ions, the 17 000-dalton fragment is further degraded and attack of the subfragment 1 link ensues. Mn(II) bound to cardiac myosin gives an EPR spectrum basically similar to that of skeletal myosin, suggesting that their 19 000-dalton light chains are analogous with respect to their divalent metal binding sites, despite their chemical differences. The potential of EPR spectroscopy for characterizing the metal binding sites of myosin from different sources and of intact muscle fibers is discussed.