Ketohexokinase (fructokinase, KHK) catalyses the phosphorylation of fructose to fructose-l-phosphate. It thereby initiates the intracellular catabolism of a large proportion of dietary carbohydrate. Although found at high level in liver, renal cortex and small intestine, fructokinase activity has also been known for many years to be present at lower levels in most other tissues. We previously found that there appeared to be two isoforms of human KHK, and have now investigated the molecular basis for this in human, rat and mouse. Cloning of the human KHK gene, on chromosome 2p23.2-2p23.3, shows that it has nine exons, spanning 14 kb. An intragenic duplication has resulted in two similar 135-bp exons (designated 3a and 3c), separated by a short intron. Exon 3a and exon 3c are mutually exclusively spliced into KHK mRNA. This exon-intron structure and the pattern of alternative splicing are conserved in both the rat and mouse, suggesting distinct conserved functions for the two KHK isoforms. The alternative splicing is also tissue specific, since in both rat and human, tissues expressing high levels of KHK (liver, kidney and duodenum) utilise exclusively the 3c exon, while other tissues use only 3a. Furthermore, comparison of human foetal and adult tissues indicates a developmental splicing shift from use of exon 3a to exon 3c.