The C2 domain acts as a membrane‐targeting module in a diverse group of proteins including classical protein kinase Cs (PKCs), where it plays an essential role in activation via calcium‐dependent interactions with phosphatidylserine. The three‐dimensional structures of the Ca2+‐bound forms of the PKCα‐C2 domain both in the absence and presence of 1,2‐dicaproyl‐ sn ‐phosphatidyl‐L‐serine have now been determined by X‐ray crystallography at 2.4 and 2.6 Å resolution, respectively. In the structure of the C2 ternary complex, the glycerophosphoserine moiety of the phospholipid adopts a quasi‐cyclic conformation, with the phosphoryl group directly coordinated to one of the Ca2+ ions. Specific recognition of the phosphatidylserine is reinforced by additional hydrogen bonds and hydrophobic interactions with protein residues in the vicinity of the Ca2+ binding region. The central feature of the PKCα‐C2 domain structure is an eight‐stranded, anti‐parallel β‐barrel with a molecular topology and organization of the Ca2+ binding region closely related to that found in PKCβ‐C2, although only two Ca2+ ions have been located bound to the PKCα‐C2 domain. The structural information provided by these results suggests a membrane binding mechanism of the PKCα‐C2 domain in which calcium ions directly mediate the phosphatidylserine recognition while the calcium binding region 3 might penetrate into the phospholipid bilayer.