Microplasmodia of Physarum polycephalum have been investigated by conventional electrophysiological techniques. In standard medium (30 mM K+, 4 mM Ca++, 3 mM Mg++, 18 mM citrate buffer, pH 4.7, 22 degrees C), the transmembrane potential difference Vm is around -100 mV and the membrane resistance about 0.25 omega m2. Vm is insensitive to light and changes of the Na+/K+ ratio in the medium. Without bivalent cations in the medium and/or in presence of metabolic inhibitors (CCCP, CN-, N3-), Vm drops to about 0 mV. Under normal conditions, Vm is very sensitive to external pH (pH0), displaying an almost Nernstian slope at pH0 = 3. However, when measured during metabolic inhibition, Vm shows no sensitivity to pH0 over the range 3 to 6, only rising (about 50 mV/pH) at pH0 = 6. Addition of glucose or sucrose (but not mannitol or sorbitol) causes rapid depolarization, which partially recovers over the next few minutes. Half-maximal peak depolarization (25 mV with glucose) was achieved with 1 mM of the sugar. Sugar-induced depolarization was insensitive to pH0. The results are discussed on the basis of Class-I models of charge transport across biomembranes (Hansen, Gradmann, Sanders and Slayman, 1981, J. Membrane Biol. 63:165-190). Three transport systems are characterized: 1) An electrogenic H+ extrusion pump with a stoichiometry of 2 H+ per metabolic energy equivalent. The deprotonated form of the pump seems to be negatively charged. 2) In addition to the passive K+ pathways, there is a passive H+ transport system; here the protonated form seems to be positively charged. 3) A tentative H+-sugar cotransport system operates far from thermodynamic equilibrium, carrying negative charge in its deprotonated states.