Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important signaling lipid that provides spatiotemporal control for a broad range of signaling events. PI(4,5)P2 is a highly charged lipid, and electrostatics play a crucial role in protein/PI(4,5)P2 interactions. The exact charge on PI(4,5)P2 can be strongly affected by cellular conditions. We have used solid-state MAS 31P-NMR to examine the ionization behavior of PI(4,5)P2 in multilamellar vesicles containing mixtures of other lipids, for pH values from 4 to 10. Previous research has shown a complex ionization pattern for PI(4,5)P2 in binary lipid mixtures of PC and PI(4,5)P2 (Kooijman et al. Biochemistry 48 (2009) 9360). In more complicated lipid mixtures, we observe significant deviations from the binary PC/PI(4,5)P2 titration curve. The hydrogen-bond donor lipid PE causes a large shift of the titration curve of the 4- and 5-phosphate of PI(4,5)P2to lower pH values, indicating a stabilization of a more negatively charged form of PI(4,5)P2. For mixtures of PC, PS, and PI(4,5)P2, the titration curve appears to show a shift to higher pH values, indicating a decrease in negative charge for PI(4,5)P2. This effect is pH dependent, with the strongest shift occurring in the physiological pH range. In the presence of PI two opposing effects lead to a net charge that is similar to the charge found for PI(4,5)P2 in the absence of PI. The enhanced negative charge in the membrane due to the presence of PI leads to an increased PI(4,5)P2 protonation (reduced charge). This effect is opposed by PI/PI(4,5)P2 hydrogen bond formation which results in increased deprotonation of the phosphomonoester groups. As a result, PI appears to have a minor effect on PI(4,5)P2 ionization, however, fluorescence microscopy measurements of PC/PI/PI(4,5)P2 GUVs show a pronounced effect on PI(4,5)P2 morphology.