The C2 domain is a Ca(2+)-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. To understand the mechanisms by which the C2 domain mediates the membrane targeting of PLC-delta isoforms, we measured the in vitro membrane binding of the C2 domains of PLC-delta1, -delta3, and -delta4 by surface plasmon resonance and monolayer techniques and their subcellular localization by time-lapse confocal microscopy. The membrane binding of the PLC-delta1-C2 is driven by nonspecific electrostatic interactions between the Ca(2+)-induced cationic surface of protein and the anionic membrane and specific interactions involving Ca(2+), Asn(647), and phosphatidylserine (PS). The PS selectivity of PLC-delta1-C2 governs its specific Ca(2+)-dependent subcellular targeting to the plasma membrane. The membrane binding of the PLC-delta3-C2 also involves Ca(2+)-induced nonspecific electrostatic interactions and PS coordination, and the latter leads to specific subcellular targeting to the plasma membrane. In contrast to PLC-delta1-C2 and PLC-delta3-C2, PLC-delta4-C2 has significant Ca(2+)-independent membrane affinity and no PS selectivity due to the presence of cationic residues in the Ca(2+)-binding loops and the substitution of Ser for the Ca(2+)-coordinating Asp in position 717. Consequently, PLC-delta4-C2 exhibits unique pre-localization to the plasma membrane prior to Ca(2+) import and non-selective Ca(2+)-mediated targeting to various cellular membranes, suggesting that PLC-delta4 might have a novel regulatory mechanism. Together, these results establish the C2 domains of PLC-delta isoforms as Ca(2+)-dependent membrane targeting domains that have distinct membrane binding properties that control their subcellular localization behaviors.