RT-PCR analysis revealed expression of mRNAs for particular GluRs, Glu transporters and vesicular Glu transporter in primary cultures of rat calvarial osteoblasts under premature to mature states. Sustained exposure to the NMDA receptor antagonist MK-801 significantly prevented increases in alkaline phosphatase activity, Ca2+ accumulation and DNA binding activity of CBFA1 in a concentration dependent manner in osteoblasts cultured for 7 to 28 DIV, without significantly affecting cell survivability. The agonist for group III mGluR L-AP4 significantly inhibited the accumulation of cAMP induced by parathyroid hormone in osteoblasts, which occurred in a manner sensitive to prevention by the group III antagonist CPPG. AMPA significantly increased the release of endogenous Glu from osteoblasts in the presence of the inhibitor of AMPA receptor desensitization cyclothiazide. The release evoked by AMPA was significantly prevented by the addition of an AMPA receptor antagonist as well as the removal of Ca2+ ions. [3H]Glu uptake was also seen in a temperature- and sodium-dependent manner in cultured osteoblasts. These results suggest that Glu may at least in part play a role in mechanisms associated with cellular proliferation and/or differentiation through particular GluR and Glu transporters functionally expressed in rat calvarial osteoblasts.