Hexokinase catalyses the first step in the metabolism of glucose, but has also been proposed to be involved in sugar sensing and signalling in both yeasts and plants. The substrates of hexokinase such as glucose (Glc) and fructose (Fruc) are also the most important sugars during grape berry ripening. The grapevine proteome database was analysed to investigate the roles of hexokinases in grape berry growth and development. Six hexokinase genes displaying high nucleotide sequence identity (72 - 87%) with hexokinase genes in other species were identified. Most of the Vitis vinifera hexokinase (VvHXK) genes had a highly conserved genomic structure consisting of nine exons and eight introns. A search for cis-regulatory elements in the promoter regions of all six hexokinase genes revealed that most were probably regulated by light, sugar, phytohormones, or abiotic stress. The isolation and expression of a hexokinase cDNA from V vinifera 'Sultanine', named VvHXK3, was also reported. Analysis of the predicted amino acid sequence of VvHXK3 suggested that the protein could be a chloroplast-stromal hexokinase with a possible transit peptide cleavage site after amino acid residue 26. The VvHXK3 gene was differentially expressed in a variety of organs including berries, leaves, roots, and pollen, but its expression was highest in berries during their early development and at the start of ripening. To determine its function, VvHXK3 cDNA was expressed in a triple mutant yeast strain that lacked the ability to phosphorylate Glc and Fru and, therefore, was unable to grow on these sugars as sole carbon source. Mutant yeast cells that expressed VvHXK3 grew on both Glc and Fru, indicating that VvHXK3 could complement this mutant and had hexokinase activity.