Calmodulin is an important second messenger protein which is involved in a large variety of cellular pathways. Calmodulin is sensitive to fluctuations in the intracellular Ca2+ levels and is activated by the binding of four Ca2+ ions. In spite of the important role it plays in signal transduction pathways, it shows a surprisingly broad specificity for binding metal ions. Using 15N-Gly biosynthetically-labelled calmodulin, we have studied the binding of different metal ions to calmodulin, including K+, Na+, Ca2+, Mg2+, Zn2+, Cd2+, Pb2+, Hg2+, Sr2+, La3+ and Lu3+, by 1H,15N HMQC NMR experiments. The effects of these ions on the substrate-binding ability of calmodulin have also been studied by fluorescence spectroscopy of the single tryptophan residue in a 22-residue synthetic peptide encompassing the skeletal muscle myosin light chain kinase calmodulin-binding domain. Most of these metal ions can activate a calmodulin target enzyme to some extent, though they bind to calmodulin in a different manner. Mg2+, which is of direct physiological interest, has a distinct site-preference for calmodulin, as it shows the highest affinity for site I in the N-terminal domain, while the C-terminal sites III and IV are the high affinity binding sites for Ca2+ (as well as for Cd2+). At a high concentration of Mg2+ and a low concentration of Ca2+, calmodulin can bind Mg2+ in its N-terminal lobe while the C-terminal domain is occupied by Ca2+; this species could exist in resting cells in which the Mg2+ level significantly exceeds that of Ca2+. Moreover, our data suggest that the toxicity of Pb(2+)--which, like Sr2+, binds with an equal and high affinity to all four sites--may be related to its capacity to tightly bind and improperly activate calmodulin.