A method was developed for determining pH differences across cell membranes using a methylammonium-selective membrane electrode, based on monitoring of the pH gradient-induced uptake of methylammonium in situ. The methylammonium electrode was constructed using calix[6]arene–hexaacetic acid hexaethyl ester as a neutral carrier and bis(2-ethylhexyl) sebacate as a membrane solvent in a poly(vinyl chloride) membrane matrix. This electrode exhibited a near-Nernstian response to methylammonium in the concentration range 2 × 10^–5–1 × 10^–2M with a slope of 58 mV per concentration decade in a buffer solution of 150 mM choline chloride–10 mM TRIS–HCl (pH 7.5). The limit of detection was 5 × 10^–6M. In experiments using liposomes, the uptake of methylammonium into liposomes occurred effectively when the pH of the outside suspension medium was alkaline, and the determination of changes in methylammonium concentrations in the outer medium was quantitatively related to changes in the pH differences across the liposomal membrane. The transmembrane pH differences in Escherichia coli cells were also determined by this method.