Based on studies of hypophosphatasia, which is a systemic skeletal disorder resulting from tissuenonspecific alkaline phosphatase (TNSALP) deficiency, TNSALP was suggested to be indispensable for bone mineralization. Recently, we demonstrated that there was a significant difference in bone mineral density (BMD) among haplotypes, which was lowest among TNSALP (787T [Tyr-246Tyr]) homozygotes, highest among TNSALP (787T > C [Tyr246His]) homozygotes, and intermediate among heterozygotes. To analyze protein translated from the TNSALP gene 787T > C, we performed the biosynthesis of TNSALPs using TNSALP cDNA expression vectors. TNSALP (787T) and TNSALP (787T > C) were synthesized similarly as a high-mannose-type 66-kDa form, becoming an 80-kDa form. Expression of the human 787T > C TNSALP gene using the cultured mouse marrow stromal cell line ST2 demonstrated that the protein translated from 787T > C exhibited an ALP-specific activity similarly to that of 787T. Interestingly, the Km value for TNSALP in ST2 cells transfected with the 787T > C TNSALP gene was decreased significantly compared to that of cells carrying the 787T gene (P < 0.01). These results suggest that the significant difference in Km values between the proteins translated from 787T > C and 787T may contribute to regulatory effects on bone metabolism.