Phosphatidylserine (PtdSer) potentiates histamine secretion from mast cells exposed to concanavalin A and Ca2+. In order to identify the form of PtdSer that is responsible for its effect on mast cell secretion, PtdSer containing a tritium-labeled serine moiety (3H-PtdSer) was synthesized from egg yolk phosphatidylcholine. The critical micele concentration (CMC) of 3H-PtdSer and the binding isotherm for 3H-PtdSer interaction with mast cells were determined. The midpoints of the binding isotherm and the dose-response curve for potentiation of secretion coincide and are 2 orders of magnitude greater than the CMC. The shape of the binding curve is explicable either in terms of simple binding of preformed PtdSer micelles or of cooperative binding of monomeric PtdSer in which the number of molecules cooperatively associating with a mast cell binding site is equal to the number of monomers in a PtdSer micelle. In either case, at equilibrium, PtdSer micelles are bound to the mast cells. The number of PtdSer molecules bound to a single mast cell at equilibrium was estimated to be 3.7 X l0s.The secretion of histamine from mast cells exposed to dextran (1), antigen (2, 3) or concanavalin A (Con A) (4, 5) is potentiated by phosphatidylserine (PtdSer). Mast cell secretion induced by these secretagogues is not potentiated by other phospholipids such as phosphatidylcholine (PtdCho), phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, or phosphatidic acid (1, 4, 6, 7). Although phospholipids have been shown to activate membrane-associated enzyme preparations after delipidization (8) as well as such processes as activation of the intrinsic (9) and extrinsic (10) coagulation pathways, to our knowledge the potentiation of mast cell secretion by PtdSer represents the only example of an in situ cellular event that has been shown to be activated by exposure of cells to a specific exogenous phospholipid.As an amphiphilic molecule, PtdSer is capable of undergoing a highly cooperative self-association in which its nonpolar fatty acyl chains are transferred from an aqueous to a hydrocarbon environment. The concentration at which self-association occurs is defined as the critical micelle concentration (CMC) (11). Micellar aggregates,* the geometry of which is determined by the length and number of hydrocarbon chains as well as the polar head group (12), predominate at total amphiphile concentrations in excess of the CMC. It is generally assumed that the CMC of biological phospholipids is sufficiently low (-10-10 M) as to make the contribution of the monomeric form negligible under most experimental conditions. Actually the CMC has been experimentally determined for only one phospholipid class, a homologous series of PtdChos containing saturated hydrocarbon chains (13,14). Precaution must be exercised in extrapolating the data for disaturated PtdCho to anionic phospholipids isolated from tissues. Naturally occurring phospholipids usually contain a large proportion of cis-unsaturated fatty acids and are not homogen...