The independently folding C2 domain motif serves as a Ca2+-dependent membrane docking trigger in a large number of Ca2+ signaling pathways. A comparison was initiated between three closely related C2 domains from the conventional protein kinase C subfamily (cPKC, isoforms α, β, and γ). The results reveal that these C2 domain isoforms exhibit some similarities but are specialized in important ways, including different Ca2+ stoichiometries. In the absence of membranes, Ca2+ affinities of the isolated C2 domains are similar (2-fold difference) while Hill coefficients reveal cooperative Ca2+ binding for the PKCβ C2 domain but not for the PKCα or PKCγ C2 domain (H = 2.3 ± 0.1 for PKCβ, 0.9 ± 0.1 for PKCα, and 0.9 ± 0.1 for PKCγ). When phosphatidylserine-containing membranes are present, Ca2+ affinities range from the sub-micromolar to the micromolar (7-fold difference) ([Ca2+]1/2 = 0.7 ± 0.1 μM for PKCγ, 1.4 ± 0.1 μM for PKCα, and 5.0 ± 0.2 μM for PKCβ), and cooperative Ca2+ binding is observed for all three C...