Hypophosphatasia (HOPS) is a heritable disorder characterized by defective skeletal mineralization, deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity and premature loss of deciduous teeth. In a previous study, we detected missense mutations in the TNSALP gene of a patient who inherited the F310L and the V365I mutation with severe periodontitis and childhood HOPS. Expression of the mutant V365I TNSALP gene using COS-1 cells demonstrated that the protein translated from the mutant had undetectable ALP activity. In the present study, we characterized another ALP enzyme translated from the mutant F310L and compared it with the ALP in the patient's serum. The COS-1 cells transfected with the F310L and co-transfected with F310L and V365I (F310L/V365I) exhibited levels of 67% and 31%, respectively, with the enzymatic activity of the wild-type taken as 100%. In the thermostability test, TNSALPs in the COS-1 cells transfected with the mutant F310L or F310L/V365I were significantly more heat labile compared with that of the wild-type. Moreover, ALP from the patient's serum was also more heat labile than normal ALP. These results suggest that the protein translated from the mutant F310L, in addition to the mutant V365I, may be responsible for the expression of symptoms of the childhood-type HOPS.