D-Gluconate uptake was studied in whole cells of Arthrobacter pyridinolis; the uptake activity was inducible, mutable and showed saturation kinetics (Km = 5 micrometer). Uptake of D-gluconate was not mediated by a phosphoenol-pyruvate : hexose phosphotransferase system, nor was it directly energized by ATP. A transmembrane pH gradient, delta pH, of --63 mV was generated by A. pyridinolis cells at pH 6.5, while at pH 7.5, delta pH = 0. Addition of 8 micrometer D-gluconate significantly reduced the delta pH. The transmembrane electrical potential, delta psi, which was --87 mV over a range of pH from 5.5 to 7.5, was unaffected by the presence of substrate. D-Gluconate accumulated at the same rate and as the protonated solute, at both pH 6.5 and 7.5. Experiments in which a diffusion potential was generated in cyanide-treated cells, indicated that the delta psi did not energize transport. Rather, the rate of D-gluconate uptake metabolism: (a) treatment of cells with valinomycin or nigericin, under conditions in which there was a loss of intracellular potassium, inhibited both D-gluconate uptake and the metabolism of pre-accumulated D-gluconate; (b) the effects of cyanide and azide on D-gluconate uptake were much more severe at pH 6.5 than pH 7.5, a pattern which paralleled the effects of these inhibitors on D-gluconate metabolism; (c) extraction and chromatography of intracellular label from D-gluconate uptake revealed that accumulation of unaltered D-gluconate was negligible; (d) a series of mutant strains with lower D-gluconate kinase activities also exhibited low rates of D-gluconate uptake; (e) spontaneous revertants of these mutant strains consistently regained both D-gluconate kinase activity and wild type levels of uptake.