Upon induced variation of membrane lipid acyl chain unsaturation in Acholeplasma laidlawii, the cells strongly change in a characteristic manner the proportions of individual (charged and noncharged) polar lipids synthesized. Monolayer analysis of polar lipid extracts revealed different mean lateral molecular areas but similar surface charge densities. Microelectrophoresis of these lipids indicated an almost constant lipid membrane zeta-potential of about -35 mV. Simulation by the Gouy-Chapman-Stern (GCS) relations verified that the zeta (surface)-potentials remain constant. Exposing cells to increasing concentration of Na+ yielded a substantial increase in amounts of charged lipids synthesized. In model systems consisting of mixtures of A. laidlawii phosphatidylglycerol (anionic) and glucolipid (diglucosyldiglyceride, noncharged) microelectrophoresis showed; (i) increasing PG amounts resulted in an increased-, and (ii) increasing Na+ concentration resulted in a decreased zeta-potential, respectively, (iii) at physiological ionic strength and lipid surface charge densities the zeta-potential was approximately -35 mV, and (iv) simulation according to the GCS theory yielded an acceptable fit with experimental data. This behavior of the phosphatidylglycerol/diglucosyldiglyceride mixtures is very similar to that of phosphatidylserine/phosphatidylcholine mixtures. It is concluded that the changes in lipid surface charge densities (and surface potential) in A. laidlawii membranes brought by variation in lateral areas of lipid acyl chains and the concentration-dependent quenching of lipid charge by Na+, is compensated for by the cellular regulation of charged lipid amounts thereby maintaining a constant lipid surface potential.