Abstract: The yeast HXT (glucose transporter gene) repressor Rgt1 lacks a dimerization domain and thus appears to bind as a monomer to its consensus binding site sequence (5'-CGGANNA-3'). The HXT1 promoter contains 8 Rgt1-binding sites, but its expression is not effectively repressed by Rgt1. In the present study, the Rgt1-binding sites in the HXT1 promoter were analyzed to examine how Rgt1 mediates transcriptional repression. It is seen that Rgt1 binds the HXT1 promoter, but does not significantly mediate repression. When engineered to be multimerized without the intervening sequences between the Rgt1-binding sites, however, 4 or more Rgt1-binding sites were required to provide sufficient Rgt1-dependent repression. These findings suggest that the intervening sequences between the Rgt1-binding sites are important for the regulation of Rgt1 function and that Rgt1 functions efficiently only through multiple binding sites.