Hexokinases catalyse the first step in glucose metabolism and play a role in glucose sensing in mammals, plants and fungi. We describe a new class of hexokinases that appear to be solely regulatory in function. The Aspergillus nidulans hxkD gene (formerly named xprF) encodes a hexokinase-like protein. We constructed hxkDDelta gene disruption mutants which showed increased levels of extracellular protease in response to carbon starvation. The hxkDDelta mutations are not completely recessive, indicating that the level of the gene product is critical. Transcript levels of hxkD increase during carbon starvation and this response is not dependent on functional HxkD. A gene encoding a second atypical hexokinase (HxkC) was identified. The hxkCDelta gene disruption mutant exhibits a phenotype similar, but not identical, to hxkDDelta mutants. As with hxkD, mutations in hxkC are suppressed by loss-of-function mutations in xprG, which encodes a putative transcriptional activator involved in the response to nutrient limitation. We show that GFP-tagged HxkD was found only in nuclei suggesting a regulatory role for HxkD. GFP-tagged HxkC was associated with mitochondria. Homologs of hxkC and hxkD are conserved in multi-cellular fungi. Genes encoding atypical hexokinases are present in many genome sequence databases. Thus, non-catalytic hexokinases may be widespread.