C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca 2+ -binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKCα-C2 domain in complex with Ca 2+ , 1,2-dihexanoyl- sn -glycero-3-[phospho- l -serine] (PtdSer), and 1,2-diayl- sn -glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns( 4 , 5 )P 2 ] shows that PtdSer binds specifically to the calcium-binding region, whereas PtdIns( 4 , 5 )P 2 occupies the concave surface of strands β3 and β4. Strikingly, the structure reveals a PtdIns( 4 , 5 )P 2 -C2 domain-binding mode in which the aromatic residues Tyr-195 and Trp-245 establish direct interactions with the phosphate moieties of the inositol ring. Mutations that abrogate Tyr-195 and Trp-245 recognition of PtdIns( 4 , 5 )P 2 severely impaired the ability of PKCα to localize to the plasma membrane. Notably, these residues are highly conserved among C2 domains of topology I, and a general mechanism of C2 domain-membrane docking mediated by PtdIns( 4 , 5 )P 2 is presented.