Addition of rapidly fermentable sugars to cells of the yeast Saccharomyces cerevisiae grown on nonfermentable carbon sources causes a variety of short-term and long-term regulatory effects, leading to an adaptation to fermentative metabolism. One important feature of this metabolic switch is the occurrence of extensive transcriptional repression of a large group of genes. We have investigated transcriptional regulation of the SUC2 gene encoding repressible invertase, and of HXK1, HXK2 and GLK1 encoding the three known yeast hexose kinases during transition from derepressed to repressed growth conditions. Comparing yeast strains that express various combinations of the hexose kinase genes, we have determined the importance of each of these kinases for establishing the catabolite-repressed state. We show that catabolite repression involves two distinct mechanisms. An initial rapid response is mediated through any kinase, including Glk1, which is able to phosphorylate the available sugar. In contrast, long-term repression specifically requires Hxk2 on glucose and either Hxk1 or Hxk2 on fructose. Both HXK1 and GLK1 are repressed upon addition of glucose or fructose. However, fructose repression of Hxk1 is only transient, which is in line with its preference for fructose as substrate and its requirement for long-term fructose repression. In addition, expression of HXK1 and GLK1 is regulated through cAMP-dependent protein kinase. These results indicate that sugar sensing and establishment of catabolite repression are controlled by an interregulatory network, involving all three yeast sugar kinases and the Ras-cAMP pathway.