We measured the electrostatic potential 1 nm from the surface of charged phospholipid bilayer membranes to test the predictions of the Gouy-Chapman theory. Fluorescent probes (anthraniloyl, 5-(dimethylamino)naphthalene-1-sulfonyl, Lucifer yellow) were attached covalently to the sialic acid residue of the ganglioside galactosyl-N-acetylgalactosaminyl(N-acetylneuraminyl)galactosylglucosylc eramide (GM1). These fluorescent gangliosides were incorporated into neutral [phosphatidylcholine (PC)] or charged [phosphatidylserine (PS)] phospholipid bilayers, and the fluorescence was quenched with the cations thallium and 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (tempamine). We calculated the electrostatic potential at the chromophore from the quenching ratio using the Boltzmann relation: the average potential was -30 mV for PS bilayers in 0.1 M NaNO3. We assume the chromophore is 1 nm from the surface because X-ray diffraction measurements demonstrate that the sialic acid residue of GM1 is 1 nm from the surface of a PC/GM1 bilayer [McDaniel, R. V., & McIntosh, T. J. (1986) Biophys. J. 49, 94-96]. We also used thallium and tempamine to quench the fluorescence of chromophores located at the surface of the PS membranes; in 0.1 M NaNO3 the average surface potential was -80 mV, which agrees with other measurements. The Gouy-Chapman theory predicts that the potential 1 nm from a membrane with a surface potential of -80 mV is -24 mV; this prediction agrees qualitatively with the experimental results obtained with fluorescent gangliosides.