A specific spectrophotometric method for the assay of fructokinase (ketohexokinase) that can be used with tissue extracts has been developed. It is based on the use as auxiliary enzymes of the recently discovered 1-phosphofructokinase, aldolase, triosephosphate isomerase and glycerolphosphate dehydrogenase, to couple fructose 1-phosphate formation to NADH2 oxidation. With this method a Michaelis constant of liver fructokinase for fructose of 0.1 mM has been obtained, and the inhibition by ADP has been characterized as largely competitive with ATP. The occurrence of fructokinase has been confirmed in kidney and intestinal mucosa, but not in adipose tissue. Some quantitative information on the specificity and reversibility of the microbial 1-phosphofructokinase is also reported. This enzyme permits a specific determination of fructose 1-phosphate in liver extracts.