Intracellular microelectrodes were used to measure the effects of glucose transport on membrane voltage and membrane resistance in Neurospora crassa. Sudden activation of glucose uptake, via the high-affinity, derepressible system II, results in a very large depolarization of the plasma membrane. At saturating concentrations of glucose, the depolarization averages 120 mV; it is diphasic in time, with an initial shift at rates of 100-200 mV/sec followed by a slow, spontaneous, partial repolarization. Changes in intracellular ATP concentration are small and could account for only 10 mV of the initial depolarization, while the rest appears to depend upon the transport process itself. A plot of peak depolarization against the extracellular glucose concentration gives a saturation curve which is half-maximal at 42 μM, in good agreement with the K1/2 reported for glucose transport via system II. The nonmetabolized analogue 3-O-methyl-D-glucose also causes depolarization, and in addition leads to a pulsed alkalinization of the medium occurring at approximately the same rate as 3-O-methyl-D-glucose uptake. The membrane resistance falls only slightly during glucose depolarization, a fact which requires the transport system itself to have a high internal resistance, or the membrane current-voltage relationship in glucose-starved cells to be quite nonlinear. All of the data support Mitchell's notion that sugar and hydrogen ions are contransported under the influence of the membrane potential, and lead to values for H+:glucose stoichiometry of 0.8 to 1.4.