In the compacted multilayered myelin sheath of the central nervous system, myelin basic protein (MBP) is thought to be responsible for adhesion of the intracellular surfaces by electrostatic interactions with acidic lipids. Noncompacted regions of myelin containing cytosol exist and can take up potassium released into the extracellular fluid after the axonal action potential. Therefore, the effect of K+ concentration on the ability of MBP to aggregate large unilamellar vesicles (LUVs) containing phosphatidylcholine (PC) and 10-20% acidic lipid was investigated. At MBP to lipid ratios where there was an excess of acidic lipid, physiological increases in K+ concentration up to about 100 mM greatly increased MBP-mediated aggregation of the LUVs by shielding the negative charge on the vesicle surface. Thus, changes in K+ concentration during the axonal action potential could regulate MBP-mediated adhesion of the intracellular myelin surfaces of noncompacted regions of myelin such as the paranodal loops. It could thus regulate the volume of these cytosolic regions, allowing MBP to have a dynamic function in myelin. Concentrations of K+ above 150 mM caused dissociation of MBP from LUVs containing PC and a single acidic lipid. LUVs containing the lipid composition estimated to be characteristic of the cytoplasmic leaflet of myelin (Cyt.-LUVs) were found to interact uniquely with MBP, resulting in greater aggregation, greater sensitivity to K+ concentration, and resistance to dissociation at high K+ concentrations. The latter suggested that electrostatic interactions were not the only force involved in binding of MBP to the Cyt.-LUVs. Hydrogen bonding of the protein to the lipid head groups and hydrophobic interactions due to penetration of hydrophobic amino acid side chains into the bilayer could also occur. The greater involvement of hydrophobic interactions of MBP with Cyt.-LUVs compared to PC/acidic lipid LUVs was confirmed from greater labeling of MBP bound to Cyt.-LUVs by the hydrophobic photolabeled TID. Cholesterol and phosphatidylethanolamine together were found to be responsible for the greater MBP-mediated aggregation of Cyt.-LUVs and the greater TID labeling of MBP bound to Cyt.-LUVs compared to PC/acidic lipid LUVs. Thus, the lipid composition of the intracellular surface of myelin is well suited to allow MBP to mediate adhesion of apposing intracellular membranes and to respond in a dynamic way in some regions of myelin, such as the paranodal loops, to changes in K+ concentration resulting from nerve conduction.