Recently, substantial evidence has accumulated that the G-protein-coupled, extracellular calcium (Ca-o(2+))-sensing receptor (CaR) is expressed in bone marrow-derived cells, including osteoblasts, stromal cells, monocytes-macrophages, and osteoclast precursor cells. Our previous studies have shown that the mouse osteoblastic MC3T3-E1 cell line also expresses the CaR and exhibits mitogenic responses when exposed to various CaR agonists. In this study, in order to understand the signaling pathway(s) mediating this response, we studied the effects of CaR agonists on the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK) (Erk1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) in MC3T3-E1 cells. Raising the level of Ca-o(2+) (4.5 mM) or addition of the polycationic CaR agonists, gadolinium (Gd3+) (25 muM), neomycin (300 muM) or spermine (1 mM), each stimulated phosphorylation of both p42/44 and p38 MAPKs, but not JNK, as assessed using phospho-specific antibodies to the respective MAPKs. Furthermore, phosphorylation of p42/44 and p38 MAPK were markedly inhibited by their selective and potent inhibitors, PD98059 (50 muM) and SB203580 (10 muM), respectively. Finally, the two inhibitors suppressed [H-3]thymidine incorporation into DNA in MC3T3-E1 cells at a normal level of Ca-o(2+) (1.8 mM) as well as when stimulated by high (4.5 mM) Ca-o(2+), Gd3+, or neomycin. Thus, in mouse osteoblastic MC3T3-E1 cells, both the p42/44 and p38 MAPK cascades play pivotal roles in CaR-stimulated mitogenic responses.