The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed of l-alpha-1,2-dimyristoylphosphatidylcholine, l-alpha-1,2-dimyristoylphosphatidylglycerol and l-alpha-1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing l-alpha-1,2-dimyristoylphosphatidylglycerol; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane. PEBP affinity for negatively charged membranes is puzzling considering the previous identification of the protein as a phosphatidylethanolamine-binding protein, and suggests that the association of PEBP with phospholipid membranes is driven by a mechanism other than its binding to solubilized phosphatidylethanolamine. An explanation was suggested by its three-dimensional structure: a small cavity at the protein surface has been reported to be the binding site of the polar head of phosphatidylethanolamine, while the N-terminal and C-terminal parts of PEBP, exposed at the protein surface, appear to be involved in the interaction with membranes. To test this hypothesis, we synthesized the two PEBP terminal regions and tested them with model membranes in parallel with the whole protein. Both peptides displayed the same behaviour as whole PEBP, indicating that they could participate in the binding of PEBP to membranes. Our results strongly suggest that PEBP directly interacts with negatively charged membrane microdomains in living cells.