PTH-related protein (PTHrP), similarly to PTH, stimulates cAMP production in target tissues. However, different potencies have been observed for the two peptides in some biological assays, suggesting that cAMP-independent second messenger pathways might be involved in PTHrP signal transduction. This hypothesis was tested in the osteogenic sarcoma cell line UMR 106–01. Addition of PTHrP-(1–34) to cell, suspensions loaded with the Ca2+ indicator indo-1 produced a transient dose-dependent increase in intracellular calcium ([Ca2+]i), with a maximal effect at 2 × 10−7 M and an ED05 at about 4 × 10−8 M. The amplitude and duration of the transients were similar to those induced by equimolar concentrations of bovine PTH-(1–34) (bPTH), and the dose-responses of the two peptides completely overlapped. Both full-length peptides, PTHrP-(1–141) and bPTH-(1–84), produced effects identical to those observed with the 1–34 fragments. Homologous and heterologous desensitization to both PTHrP-(1–34) and PTHrP - (1–141) occurred when the cells were prestimulated with equimolar or 10-fold lower doses of either PTHrP-(1–34) or bPTH- (1–34). Desensitization to bPTH-(1–34) was also observed when cells were prestimulated with PTHrP-(1–34). Furthermore, pretreatment with either bPTH-(3–34) or [Nle818, Tyr34]bPTH-(3–34) amide did not affect [Ca2+]i, but reduced the response to PTHrP-(1–34) by 55 ± 10% (n = 3) and 67 ± 8% (n = 3), respectively. The PTHrP-(1–34)-induced [Ca2+]i transient was not substantially affected by either extracellular Ca2+ chelation by EGTA or pretreatment with diltiazem, and nitrendipine only partially inhibited the [Ca2+]i response to PTHrP-(1–34) by about 10%. These results indicate that in osteoblastic cells PTHrP mobilizes Ca2+ from an intracellular storage pool with potency equal to that of PTH, and that the two hormones interact with the same receptor.