Addition of rapidly fermentable sugars to cells of the yeast Saccharomyces cerevisiae grown on non-fermentable carbon sources causes a variety of shortterm and longterm 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 HXKI, HXK2 and GLKI 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 Hxkl or Hxk2 on fructose. Both HXKl and GLKI are repressed upon addition of glucose or fructose. However, fructose repression of HXKl is only transient, which is in line with its preference for fructose as substrate and its requirement for longterm fructose repression. In addition, expression of HXKI and GLKI 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.