An artificial glucokinase/phosphatase porous membrane separating two unequal compartments of a diffusion cell was used to pump glucose from one compartment against the concentration gradient of the opposite one. Our results mainly demonstrate that, a kinase/phosphatase reactional sequence acting on both parts of a pore structure and necessarily in unstirred layers may pump a neutral solute from one compartment against the concentration gradient of the opposite one without any detectable pollution of the charged intermediary product. The corresponding theoretical analysis, which underlines the key role played by the diffusion layers located on both parts of this bienzymic membrane and the membrane＇s charge effect, was found in good agreement with the experimental data. This study corroborates well the new kinetic model recently proposed for primary scalar active transport of small hydrophilic molecules involving ATP as the energy supply.