Lead is a potent environmental toxin that mimics the effects of divalent metal ions, such as zinc and calcium, in the context of specific molecular targets and signaling processes. The molecular mechanism of lead toxicity remains poorly understood. The objective of this work was to characterize the effect of Pb2+ on the structure and membrane-binding properties of C2 alpha. C2 alpha is a peripheral membrane-binding domain of Protein Kinase c alpha (PKC alpha), which is a well-documented molecular target of lead. Using NMR and isothermal titration calorimetry (ITC) techniques, we established that C2 alpha binds Pb2+ with higher affinity than its natural cofactor, Ca2+. To gain insight into the coordination geometry of protein-bound Pb2+, we determined the crystal structures of apo and Pb2+-bound C2 alpha at 1.9 and 1.5 angstrom resolution, respectively. A comparison of these structures revealed that the metal-binding site is not preorganized and that rotation of the oxygen-donating side chains is required for the metal coordination to occur. Remarkably, we found that holodirected and hemidirected coordination geometries for the two Pb2+ ions coexist within a single protein molecule. Using protein-to-membrane Forster resonance energy transfer (FRET) spectroscopy, we demonstrated that Pb2+ displaces Ca2+ from C2 alpha in the presence of lipid membranes through the high-affinity interaction with the membrane-unbound C2 alpha. In addition, Pb2+ associates with phosphatidylserine-containing membranes and thereby competes with C2 alpha for the membrane-binding sites. This process can contribute to the inhibitory effect of Pb2+ on the PKCa activity.