The influence of transmembrane ion fluxes on mycoplasma membrane potentials was studied. Fluorescence intensity of potential-sensitive carbocyanine dyes was calibrated vs. electric membrane potential. Potassium and sodium ion diffusion potentials significantly contributed to mycoplasma membrane potential. Chloride ions were obviously freely permeable across mycoplasma membranes. Under growth conditions the mycoplasma membrane potential was estimated to be delta psi = - 80 mV.