An in vitro system was established to measure secondary active transport mediated by plant H+ symporters. For this purpose plasma membranes of Schizosaccharomyces pombe cells transformed with the HUP1 gene coding for the H+/hexose symporter of Chlorella kessleri were fused with cytochrome-c oxidase containing proteoliposomes. After energization with ascorbate/TMPD/cytochrome c these vesicles built up a protonmotive force of > 130 mV consisting mainly of a membrane potential of > 100 mV (inside negative). Energized vesicles accumulated D-glucose in a pH-dependent way up to 30-fold which was not the case with control vesicles prepared from cells transformed with the plasmid not containing the HUP1 gene. The Km value for D-glucose uptake was 5 x 10(-5) M. The pH-dependence of accumulation was not due to a difference in protonmotive force, but reflected the pH-dependence of the carrier activity, i.e., the accumulation was determined by kinetic and by thermodynamic parameters. In the system both components of protonmotive force delta psi and delta pH can be manipulated individually, which allows to evaluate to what extent they contribute to sugar accumulation. The results indicate that under certain conditions the internal pH may be a limiting factor for D-glucose accumulation.