Rat liver ketohexokinase (ATP:D-fructose 1-phosphotransferase; EC 220.127.116.11) was purified to homogeneity and the molecular mass of the protein was found by mass spectrometry to be 32,800 Da. The enzyme was cleaved and the amino acid sequences of seven peptides, comprising 24% of the total sequence, were determined. This sequence information was used to design oligonucleotide primers for a PCR using rat liver single-stranded cDNA as a template. The 224 bp PCR product was used as a probe to screen a rat liver cDNA library. A cDNA sequence of 1342 bp was obtained from three positive clones. This contained the entire coding region for ketohexokinase, and all seven peptides were identified in the predicted amino acid sequence. When ketohexokinase was expressed in Saccharomyces cerevisiae using the yeast expression vector pMA91, the cells became intolerant of the presence of fructose in their growth media. The growth of an exponential-phase culture was completely arrested within 90 min by the addition of fructose to a final concentration as low as 0.1% (w/v). This response is associated with an accumulation of fructose 1-phosphate. The cDNA for ketohexokinase encodes a protein composed of 299 amino acids with a combined molecular mass of 32,728 Da. This is in close agreement with the value for the isolated protein determined by mass spectrometry. The primary structure does not show any significant homology with those of other eukaryotic hexokinases, but it contains a highly conserved region that is present in three prokaryotic phosphotransferases that have furanose substrates.