To determine the mechanism of calcium-dependent membrane binding of cytosolic phospholipase A2 (cPLA2), we measured the interactions of cPLA2 with phospholipid monolayers and polymerizable mixed liposomes containing various phospholipids. In the presence of calcium, cPLA2 showed much higher penetrating power than secretory human pancreatic PLA2 toward anionic and electrically neutral phospholipid monolayers. cPLA2 also showed ca. 30-fold higher binding affinity for nonpolymerized 2, 3-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-1-phosphoglycerol (D-BLPG) liposomes than for polymerized ones where the membrane penetration of protein is significantly restricted. Consistent with this difference in membrane binding affinity, cPLA2 showed 20-fold higher activity toward fluorogenic substrates, 1-O-(1-pyrenedecyl)-2-arachidonoyl-sn-glycero-3-phosphocholine, inserted in nonpolymerized D-BLPG liposomes than the same substrate in polymerized D-BLPG liposomes. Furthermore, cPLA2 showed much higher sn-2 acyl group specificity (arachidonate specificity) and headgroup specificity in nonpolymerized D-BLPG liposomes than in polymerized D-BLPG liposomes. Finally, diacylglycerols, such as 1, 2-dioleoyl-sn-glycerol, selectively enhanced the membrane penetration, hydrophobic membrane binding, and interfacial enzyme activity of cPLA2. Taken together, these results indicate the following: (1) calcium not only brings cPLA2 to the membrane surface but also induces its membrane penetration. (2) This unique calcium-dependent membrane penetration of cPLA2 is necessary for its interfacial binding and substrate specificity. (3) Diacylglycerols might work as a cellular activator of cPLA2 by enhancing its membrane penetration and hydrophobic membrane binding.