Significant amounts of inorganic polyphosphates and of polyphosphate-degrading exopolyphosphatase activity were detected in human mandibular-derived osteoblast-like cells. The amount of both soluble and insoluble long-chain polyphosphate in unstimulated osteoblast-like cells was higher than in human gingival cells, erythrocytes, peripheral blood mononuclear cells, and human blood plasma. The cellular content of polyphosphate in osteoblast-like cells strongly decreased after a combined treatment of the cells with the stimulators of osteoblast proliferation and differentation, dexamethasone, β-glycerophosphate, epidermal growth factor, and ascorbic acid. The amount of soluble long-chain polyphosphate, but not the amount of insoluble long-chain polyphosphate, further decreased after an additional treatment with 1α,25-dihydroxyvitamin D3(1,25(OH)2D3). The decrease in polyphosphate content during treatment with dexamethasone, β-glycerophosphate, epidermal growth factor, and ascorbic acid was accompanied by a decrease in exopolyphosphatase, pyrophosphatase, and alkaline phosphatase activity. However, additional treatment with 1,25(OH)2D3 resulted in an increase in these enzyme activities. Osteoblast-like cell exopolyphosphatase activity and exopolyphosphatase activity in yeast, rat tissues, and human leukemia cell line HL60 were inhibited by the bisphosphonates etidronate and, to a lesser extent, clodronate and pamidronate. From our results, we assume that inorganic polyphosphate may be involved in modulation of the mineralization process in bone tissue.