Osteoblasts express protease-activated receptor-1 (PAR-1), which is activated by thrombin or by synthetic peptides corresponding to the new "tethered ligand" N-terminus of PAR-1 created by receptor cleavage. Both thrombin and human PAR-1-activating peptide stimulate an elevation of [Ca2+]i in the human SaOS-2 osteoblast-like cell line, but the peptide stimulates receptor-mediated Ca+ entry, whereas thrombin does not. Stimulation of proliferation in rat primary osteoblast-like cells is greater in response to rat PAR-1-activating peptide than to thrombin. Because the PAR-1-activating peptides are now known to activate PAR-2, the current study was undertaken to investigate whether osteoblasts express this receptor and, if so, whether this could account for the observed discrepancies between responses of osteoblasts to thrombin and to PAR-1-activating peptides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemical studies demonstrated expression of PAR-2 by primary cultures of rat calvarial osteoblast-like cells. In immunohistochemical studies of embryonic mouse bones, osteoblasts showed positive staining for the presence of PAR-2. Activators of PAR-2 include trypsin, mast cell tryptase, gingipain-R, and synthetic peptides corresponding to the PAR-2 tethered ligand sequence. Treatment of primary rat osteoblast-like cells with rat PAR-2-activating peptide (SLIGRL), or SaOS-2 cells with human PAR-2-activating peptide (SLIGKV), caused a dose-dependent increase in [Ca2+]i. Trypsin or gingipain-R also induced an increase in intracellular calcium concentration, and caused reciprocal cross desensitization. Activators of PAR-2 caused a sharp peak in [Ca2+]i followed by a sustained plateau; [Ca2+]i returned to baseline levels upon treatment with ethylene-glycol tetraacetic acid (EGTA). Treatment of rat osteoblast-like cells in vitro with SLIGRL did not affect thymidine incorporation or endogenous alkaline phosphatase activity. The results presented here demonstrate that osteoblasts express PAR-2, and that such expression is able to account for the observed discrepancies between thrombin and PAR-1-activating peptides in their ability to evoke calcium entry, but not proliferative responses.