C-2 domains are found in many proteins involved in membrane traffic or signal transduction. Although C-2 domains are thought to bind calcium ions, the structural basis for calcium binding is unclear. Analysis of calcium binding to C-2 domains of synaptotagmin I and protein kinase C-beta by nuclear magnetic resonance spectroscopy revealed a bipartite calcium-binding motif that involves the coordination of two calcium ions by five aspartate residues located on two separate loops. Sequence comparisons indicated that this may be a widely used calcium-binding motif, designated here as the C-2 motif.