A n isotope dilution method, using ³²P-labeled pyrophosphate, has been developed for the measurement of inorganic pyrophosphate (PP₁) in human plasma. The specificity of the method was better than 90% as assessed by elution patterns during ion-exchange chromatography, by paper chromatography, and by incubation with inorganic pyrophosphatase. The 99% confidence limits for a single estimation of plasma PP₁ was ±13%. There were no differences in plasma PP₁ between men and women, but the values in young people (0-15 yr) were slightly higher than in older people. The mean concentration (±SE) of PP₁ in the plasma of 73 men and women was 3.50 ±0.11 μmoles/liter (0.217 ±0.007 μg P/ml) and the normal range (99% limits) was 1.19-5.65 μmoles/liter (0.074-0.350 μg P/ml).It has been suggested that PP₁ may be important in calcium metabolism because PP₁ can prevent the precipitation of calcium phosphates in vitro and in vivo, and can slow the rates at which hydroxyapatite crystals grow and dissolve. Plasma PP₁ was therefore measured in several disorders of bone. Normal values were found in osteogenesis imperfecta, osteopetrosis, “acute” osteoporosis, and primary hyperparathyroidism. Plasma PP₁ was invariably raised in hypophosphatasia. The excess of PP₁ in plasma might be the cause of the defective mineralization in hypophosphatasia and the function of alkaline phosphatase in bone may be to act as a pyrophosphatase at sites of calcium deposition.