Abstract: Asn(331) in transmembrane segment 7 of the yeast Saccharomyces cerevisiae transporter Hxt2 has been identified as a single key residue for high-affinity glucose transport by comprehensive chimera approach. The glucose transporter GLUT1 of mammals belongs to the same major facilitator superfamily as Hxt2 and may therefore show a similar mechanism of substrate recognition. The functional role of Ile(287) in human GLUT1, which corresponds to Asn(331) in Hxt2, was studied by its replacement with each of the other 19 amino acids. The mutant transporters were individually expressed in a recently developed yeast expression system for GLUT1. Replacement of Ile(287) generated transporters with various affinities for glucose that correlated well with those of the corresponding mutants of the yeast transporter. Residues exhibiting high affinity for glucose were medium-sized, non-aromatic, uncharged and irrelevant to hydrogen-bond capability, suggesting an important role of van der Waals interaction. Sensitivity to phloretin, a specific inhibitor for the presumed exofacial glucose binding site, was decreased in two mutants, whereas that to cytochalasin B, a specific inhibitor for the presumed endofacial glucose binding site, was unchanged in the mutants. These results suggest that Ile(287) is a key residue for maintaining high glucose affinity in GLUT1 as revealed in Hxt2 and is located at or near the exofacial glucose binding site.