Enzymatic properties of F-actin ATPase [EC 3.6.1.3] at acid pH were investigated in details with a special reference to the formation of paracrystals. The pH optimum was around pH 4.7 at 45°C. The apparent activation energy was 22 kcal/mole. At pH 5.0 and 45°C, the ATPase activity was proportional to both protein concentration and incubation time, and the specific activity was approximately 0.3 μmole/mg/hr. The apparent Michaelis constant was 4×10−4 M. Ca and Mg ions inhibited the ATPase activity, while K ion enhanced it up to 0.4M. The substrate specificity was found to be so broad that ITP, UTP, CTP, GTP and deoxyATP were hydrolyzed at a similar rate to ATP. However, ADP, inorganic triphosphate and adenosine tetraphosphate were not dephosphorylated. Based on the rapid exchange reaction of the bound nucleotide with added ATP, it was concluded that ATP splitting was due to “partial interruption” of F-actin structure at acid pH. The ATP splitting action at acid pH may indicate the dynamic state of F-actin which eventually leads to the formation of the paracrystal (Type I), although the ATP splitting is not the only necessary factor.