Docking of C2 domains to target membranes is initiated by the binding of multiple Ca2+ ions to a conserved array of residues imbedded within three otherwise variable Ca2+-binding loops. We have located the membrane-docking surface on the Ca2+-activated C2 domain of cPLA2 by engineering a single cysteine substitution at 16 different locations widely distributed across the domain surface, in each case generating a unique attachment site for a fluorescein probe. The environmental sensitivity of the fluorescein-labeled cysteines enabled identification of a localized region that is perturbed by Ca2+ binding and membrane docking. Ca2+ binding to the domain altered the emission intensity of six fluoresceins in the region containing the Ca2+-binding loops, indicating that Ca2+-triggered environmental changes are localized to this region. Similarly, membrane docking increased the protonation of six fluoresceins within the Ca2+-binding loop region, indicating that these three loops also are directly involved in membrane docking. Furthermore, iodide quenching measurements revealed that membrane docking sequesters three fluorescein labeling positions, Phe35, Asn64, and Tyr96, from collisions with aqueous iodide ion. These sequestered residues are located within the identified membrane-docking region, one in each of the three Ca2+-binding loops. Finally, cysteine substitution alone was sufficient to dramatically reduce membrane affinity only at positions Phe35 and Tyr96, highlighting the importance of these two loop residues in membrane docking. Together, the results indicate that the membrane-docking surface of the C2 domain is localized to the same surface that cooperatively binds a pair of Ca2+ ions, and that the three Ca2+-binding loops themselves provide most or all of the membrane contacts. These and other results further support a general model for the membrane specificity of the C2 domain in which the variable Ca2+-binding loops provide headgroup recognition at a protein-membrane interface stabilized by multiple Ca2+ ions.