Phosphofructokinase (ATP: D-fructose 6-phosphate 1-transferase) catalyzes the phosphorylation of fructose 6-phosphate (Fru-6-P) by ATP to from fructose 1,-bisphosphate, and is considered to be the key enzyme in glycolytic pathway in liver and other tissues. In dental pulp, both aerobic and anaerobic pathways of carbohydrate metabolism are present. It was reported that oxygen consumption was parallel to the activity of dentin formation, and anaerobic pathway became predominant after the cession of calcification. Analysis of glycolytic activity in bovine dental pulp revealed that PFK was also the key enzyme in this tissue. But, the regulatory mechanism of PFK in dental pulp has not been elucidated yet. In this paper, properties and regulatory mechanism of PFK in rabbit molar teeth were examined, and the following results were obtained. 1. At nearly neutral pH, PFK activity was extremely low, and it increased with the shift of pH to more alkaline side. Maximum activity was obtained over pH 8.0. 2. At physiological pH, PFK was activated with low concentration of ATP, but was inhibited with high concentration of ATP. 3. For the recovery of PFK activity from ATP-dependent inhibition, not only Fru-6-P but also AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) were necessary. 4. In rabbit dental pulp, PFK was regulated synergistically by Fru-6-P, ATP, AMP and Fru-2,6-P2, and the range of the actual PFK activity seemed to be 30 to 80% of the maximum activity. 5. PFK was activated also with inorganic phosphate. But, addition of calcium resulted in the inhibition of PFK.