We have developed a new heterologous expression system for mammalian glucose transporters. The system is based on a Saccharomyces cerevisiae strain completely deleted for all its endogenous hexose transporters and unable to take up and to grow on hexoses. To target the heterologous glucose transporters into the yeast plasma membrane in a fully active form, additional mutations had to be introduced into the hexose transport-deficient strain. Although GLUT1 was localized at the cell surface already in the parent strain, it supported uptake of glucose only in an Δhxt fgy1-1 mutant strain. Moreover, various mutations within the first half of the second predicted transmembrane helix converted GLUT1 into a form able to support uptake of glucose into yeast cells. GLUT4 was trapped in intracellular structures but became functionally expressed in the plasma membrane in Δhxt fgy1-1 fgy4X mutant strains. Glucose transport kinetics were determined with intact yeast cells by zero-trans influx measurements with a Km of 3.2 mM for human GLUT1 and of 12.6 mM for human GLUT4. Cytochalasin B inhibited these activities. Growth tests revealed that both transporter proteins are able to mediate uptake of glucose, mannose and galactose, but not of fructose. The new heterologous expression system should be a valuable tool to develop cell based high-throughput screening assays for identifying pharmaceutical compounds influencing the transporters.