The enzymatic synthesis of fructose 1,6-diphosphate (FDF) from glucose and the enzymatic ATP regeneration were performed simultaneously in an ultrafiltration hollow-fiber reactor using both the purified enzymes and the crude cell extract of Bacillus stearothermophilus. The process consisted of the three-step synthetic reactions catalyzed by glucokinase, phosphoglucose isomerase and phosphofructokinase, and the ATP regeneration reaction catalyzed by acetate kinase. The experimental results of the yield and the recycle number agreed well with the theoretical predictions calculated using a computer program developed in our preceding study. This proved the validity of this computer program for predicting or analyzing the reactor performance.