Schacht (Schacht, J. (1976) J. Neurochem. 27, 1119-1124) demonstrated that neomycin, an aminoglycoside antibiotic, binds with high affinity to phosphatidylinositol 4,5-bisphosphate (PIP2). We investigated the binding of neomycin to PIP2 by making electrophoretic mobility measurements with multilamellar bilayer vesicles and surface potential measurements with monolayers. The bilayers and monolayers were formed from mixtures of PIP2 and egg phosphatidylcholine (PC) in 0.1 M KCl at pH 7. Neomycin does not bind to PC; 10(-3) M neomycin affects neither the zeta potential of PC vesicles nor the surface potential of PC monolayers. In contrast, 10(-6) M neomycin reduces the magnitude of the zeta potential of PC/PIP2 vesicles (5, 9, and 17 mol% PIP2) and the surface potential of monolayers (17 mol% PIP2) to less than 50% of their initial values. The electrophoretic mobility results indicate that neomycin forms an electroneutral complex with PIP2; high concentrations (greater than 10(-4) M) of neomycin reduce the zeta potential of the PC/PIP2 vesicles to zero. We could describe our data with the Gouy-Chapman-Stern theory assuming the intrinsic association constant of the 1:1 neomycin-PIP2 complex is 10(5) M-1. Neomycin is widely used in cell biology to interfere with the generation of second messengers; we discuss the relevance of our results to these studies. Specifically, 10(-6) M neomycin binds greater than 50% of the PIP2 in a bilayer or monolayer but 10(-5)-10(-3) M neomycin is required to affect the turnover of PIP2 in permeabilized platelets, mast cells, and sea urchin eggs. This result is consistent with a hypothesis that most of the PIP2 in the inner leaflet of these plasma membranes is not accessible to neomycin because it is associated with proteins.