Endothelial nitric oxide synthase (eNOS) is a calmodulin (CaM)-dependent, membrane-associated, myristoylated enzyme, which has an important role in regulation of vascular tone and platelet aggregation. In this study, wild-type and mutant forms of bovine eNOS were overexpressed in a baculovirus/Sf9 insect cell system and examined for interactions with membrane phospholipids. Purified wild-type eNOS binds to pure anionic phospholipid vesicles but not to neutral phospholipid vesicles, demonstrating that eNOS attachment to lipid bilayers requires electrostatic as well as hydrophobic interactions. Moreover, catalytic activity of the enzyme is potently inhibited by anionic phospholipids, notably phosphatidylserine (PS), but not by neutral phospholipids. eNOS activity is also significantly inhibited upon enzyme binding to biological membranes isolated from cultured cells. Binding of eNOS to PS vesicles prevents subsequent binding of the enzyme to CaM-Sepharose. Interactions of eNOS with PS are not affected by site-specific mutation of the myristic acid acceptor site in the enzyme. Deletional mutation of the eNOS CaM-binding domain, however, results in loss of binding capacity of the enzyme not only for CaM-Sepharose but also for PS vesicles. Furthermore, removal of the CaM-binding domain converts eNOS from a membrane to a cytosolic protein when the enzyme is expressed in Sf9 cells. These data demonstrate that electrostatic interactions between anionic membrane phospholipids and basic residues in the eNOS CaM-binding domain are important for enzyme membrane association. Membrane association can thus function to inhibit eNOS catalytic activity by interfering with the interaction of the enzyme with calmodulin.