Parathyroid hormone (PTH), an 84-amino-acid polypeptide, is a major systemic regulator of calcium homeostasis that activates PTH/PTHrP receptors (PTH1Rs) on target cells. Carboxyl (C) fragments of PTH, secreted by the parathyroids or generated by PTH proteolysis in the liver, circulate in blood at concentrations much higher than intact PTH(1-84) but cannot activate PTH1Rs. Receptors specific for C-PTH fragments (CPTHRs), distinct from PTH1Rs, are expressed by bone cells, especially osteocytes. Activation of CPTHRs previously was reported to modify intracellular calcium within chondrocytes. To further investigate the mechanism of action of CPTHRs in osteocytes, cytosolic free calcium ([Ca²⁺]i) was measured in the PTH1R-null osteocytic cell line OC 59, which expresses abundant CPTHRs but no PTH1Rs. [Ca²⁺]i was assessed by single-cell ratiometric microfluorimetry in Fura -2 loaded OC 59 cells. A rapid and transient increase in [Ca²⁺]i was observed in OC59 cells in response to the CPTH fragment hPTH(53-84) (250 nM). No [Ca²⁺]i signal was observed in COS-7 cells, in which CPTHR binding also cannot be detected. Neither hPTH(1-34) nor a mutant CPTH analog, [Ala^55-57]hPTH(53-84), that does not to bind to CPTHRs, increased [Ca²⁺]i in OC59 cells. The [Ca²⁺]i response to hPTH(53-84) required the presence of extracellular calcium and was blocked by inhibitors of voltage-dependent calcium channels (VDCC), including nifedipine (100 nM), -agatoxin IVA (10 nM) and -conotoxin GVIA (100 nM). We conclude that activation of CPTHRs in OC59 osteocytic cells leads to a rapid increase in influx of extracellular calcium, most likely through opening of VDCCs.