Inorganic pyrophosphate (PPi) influences the formation of bone mineral. In the rare inherited disease hypophosphatasia, abnormal extracellular metabolism of PPi occurs together with defective skeletal mineralization. The primary biochemical defect in this condition is a deficiency of the bone/liver/kidney (tissue nonspecific) isoenzyme of alkaline phosphatase (AP), an enzyme that catalyzes the extracellular breakdown of PPi. Fibroblast lines derived from patients with hypophosphatasia manifest the deficiency of AP activity that occurs in vivo and thus are a suitable model for this condition. Using these cells from patients with the severe (infantile) form of the disease, we examined aspects of PPi metabolism in hypophosphatasia, in particular the formation of PPi from ATP by ecto-nucleoside triphosphate (NTP) pyrophosphatase. This enzyme is believed to catalyze the extracellular generation of PPi in vivo. We found that normal fibroblasts possess ecto-NTP pyrophosphatase and that infantile hypophosphatasia cell lines have normal activity and cellular distribution of this enzyme compared with cell lines derived from age-matched normal subjects. This suggests that extracellular generation of PPi is normal in hypophosphatasia. The results also provide further evidence that ecto-NTP pyrophosphatase and AP are distinct entities and that hypophosphatasia does not involve a general loss of enzyme activities from cell surfaces.