Glucose is an essential nutrient, and a regulator of gene expression in eukaryotic cells. Here, a comparative, function-based genomic approach has been used to identify glucose regulatory elements and transduction pathways common to both yeast and mammalian cells. We have isolated a region in the promoter of the Saccharomyces cerevisiae hexose transporter gene HXT1 that conferred glucose sensitivity in yeast, when located upstream of the minimal CYC1 promoter. This element contained binding motifs for Rgt1, a transcriptional modulator involved in the yeast glucose-induction pathway, that were sufficient to elicit glucose responsiveness. The HXT1 regulatory element was then fused to the minimal cytomegalovirus promoter (HXT1-MIN) and inserted into an adenovirus for delivery to human fibroblasts, where it exhibited glucose-dependent transcriptional activation. Glucose action was mimicked by fructose and unrelated to glucose 6-P content, whilst non-metabolizable glucose analogues showed no effect. Activation of AMP kinase by 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranosanide blocked glucose induction, revealing parallels with the yeast glucose-repressing pathway. In contrast, delivery of Rgt1 to fibroblasts did not modify HXT1-MIN responsiveness. Thus, elements of the S.cerevisiae HXT1 gene conserve glucose regulation in human fibroblasts equivalent to the metabolism-dependent, glucose-repressing pathway in yeast. These data suggest that the instructions carried within gene regulatory elements controlling nutrient regulation of gene expression have been conserved throughout evolution.