Despite the profound physiological consequences associated with peripheral membrane protein localization, only a rudimentary understanding of the interactions of proteins with membrane surfaces exists because these questions are inaccessible by commonly used structural techniques. Here, we combine high resolution field-cycling 31 P NMR relaxation methods with spinlabeled proteins to delineate specific interactions of a bacterial phospholipase C with phospholipid vesicles. Unexpectedly, discrete binding sites for both a substrate analogue and a different phospholipid (phosphatidylcholine) known to activate the enzyme are observed. The lifetimes for the occupation of these sites (when the protein is anchored transiently to the membrane) are >1-2 s (but <1 ms), which represents the first estimate of an off-rate for a lipid dissociating from a specific site on the protein and returning to the bilayer. Furthermore, analyses of the spin-label induced NMR relaxation corroborates the presence of a discrete tyrosine-rich phosphatidylcholine binding site whose location is consistent with that suggested by modeling studies. The methodology illustrated here may be extended to a wide range of peripheral membrane proteins.Transient interactions of peripheral membrane proteins with bilayers play critical roles in signal transduction cascades. Many of these proteins are proposed to have binding interactions with multiple distinct types of phospholipids (now usually characterized by bulk protein binding with different lipid components and mutagenesis to see whether suspected regions on the protein are correlated with higher affinity binding of the protein). Confirming whether there is a discrete site, whether a lipid binds, and with what affinity, when the protein interacts with a multicomponent bilayer is difficult at best. Nonspecific interactions with the bilayer also can occur. Conformation of the bound macromolecule also is hard to deduce, particularly if the affinity of the protein for the interface is of only moderate affinity (a trait desirable in many peripheral proteins if they are to bind and dissociate from the membrane repeatedly). These proteins often have flexible loops or domains that are likely to be affected by membrane binding. Methods that can address whether and where specific phospholipid sites exist on a peripheral protein when anchored to a bilayer would aid in understanding how its function may be modulated.An interesting case is the secreted phosphatidylinositol-specific phospholipase C (PI-PLC) 2 from Bacillus thuringiensis. This small (35 kDa) enzyme is a good model for the catalytic domain of the human PI-PLC. It specifically catalyzes the cleavage of PI to form diacylglycerol and inositol 1-phosphate. Crystal structures of this protein show a monomeric ␣-barrel (1). Anionic phospholipids such as phosphatidylmethanol (PMe) are competitive inhibitors and can therefore serve as substrate analogues (2). PI-PLC is activated specifically by phosphatidylcholine (PC) present in the interface (2-4); the PC ...