Calmodulin is a ubiquitous calcium-binding protein that mediates many of the intracellular actions of Ca2+ ions. The calcium-binding sites of calmodulin consist of four EF-hand motifs; full activation of calmodulin normally occurs when all four sites are occupied by Ca2+. Inorganic lead (PY2+) has been shown to activate calmodulin at total lead concentrations similar to the concentrations of Ca2+ required for activation (Goldstein and Ar, 1983; Habermann et al., 1983), but the free Pb2+ concentrations required for calmodulin activation have not been determined. In addition, it is possible that activation may occur with different sites occupied by different divalent cations, for example Ca2+ and Pb2+. We investigated the ability of free Pb2+, alone or in combination with Ca2+, to activate calmodulin. In aqueous media, N-phenyl-1-naphthylamine (NPN) and 8-anilino-1-naphthalenesulfonate (ANS) show increased fluorescence when bound to hydrophobic regions of proteins. This increased fluorescence has been used to monitor the conformational change that occurs during calmodulin activation (LaPorte et al., 1980). In the presence of calmodulin, both Ca2+ and Pb2+ stimulated increased fluorescence of NPN and ANS. Threshold and EC50 free metal concentrations were approximately 100 nM and 450-500 nM, respectively, for Ca2+ and 100 pM and 400-550 pM, respectively, for Pb2+. Fluorescence was enhanced by combinations of low concentrations of free Ca2+ and Pb2+; for example, as little as 20 pM free Pb2+ enhanced fluorescence in combination with 200 nM free Ca2+. The activity of the PDE1 isoform of cyclic nucleotide phosphodiesterase is stimulated by Ca2+/calmodulin (Wang et al., 1990). In the presence of calmodulin, we found that Ca2+ and Pb2+ activated calmodulin-stimulated PDE activity, with threshold and EC50 free metal concentrations of approximately 200 nM and 1200 nM, respectively, for Ca2+ and 300 pM and 430 pM, respectively, for Pb2+. PDE activity was stimulated by combinations of Ca2+ and Pb2+. For example, with 100 nM free Ca2+, as little as 50 to 100 pM free Pb2+ further stimulated PDE activity; with 1000 nM free Ca2+, 20 to 50 pM free Pb2+ further stimulated PDE activity. Isobolographic analysis indicated that stimulation of PDE by Ca2+ and Pb2+ was additive. These results show that concentrations of free Pb2+ as low as 100 to 300 pM activate calmodulin and that, in the presence of physiological concentrations of free Ca2+, Pb2+ can activate calmodulin at concentrations below 50 pM. The intracellular free Ca2+ concentration in Ca2+ "hot spots," for example near sites of influx through Ca2+-permeable plasma membrane channels, can reach dozens of pM, with the free Ca2+ concentration decreasing rapidly with distance from the source of the hot spot. Our results suggest that picomolar concentrations of intracellular free Pb2+ should expand both the effective amplitude and volume of Ca2+ hot spots with respect to calmodulin activation, and thus may amplify intracellular Ca2+ signaling in lead-exposed cells.