5-Ketofructose reductase [D(-)fructose:(NADP+) 5-oxidoreductase] was purified to homogeneity from Erwinia citreus and demonstrated to catalyse the reversible NADPH-dependent reduction of 5-ketofructose (D-threo-2,5-hexodiulose) to D-fructose. The enzyme appeared as a single species upon analyses by SDS/polyacrylamide-gel electrophoresis and isoelectric focusing with an apparent relative molecular mass of 40,000 and an isoelectric point of 4.4. The amino acid composition of the enzyme and the N-terminal sequence of the first 39 residues are described. The steady-state kinetic mechanism was an ordered one with NADPH binding first to the enzyme and then to 5-ketofructose, and the order of product release was D-fructose followed by NADP+. The reversible nature of the reaction offers the possibility of using this enzyme for the determination of D-fructose.