In the present study, we investigated if Cl- regulates the proliferation of the MC3T3-E1 osteoblastic cells. The proliferation of MC3T3-E1 osteoblastic cells was diminished by lowering the extracellular Cl- concentration ([Cl-](o)) in the culture medium. The lowered in [Cl-](o) increased the periods of the G(0)/G(1) and the G(2)/M phases in cell cycle. We further studied the effects of [Cl-](o), on the key enzymes, Rb and cdc2, playing key roles in checking points of the G(0)/G(1) and the G(2)/M phases in cell cycle. The lowered in [Cl-](o) diminished the active forms of enzymes, Rb and cdc2. We further found that the action of lowered [Cl-](o) on the cell proliferation, the cell cycle, Rb and cdc2 was abolished by the presence of 2 mM glutamine, but not by that of pyruvate as another Krebs cycle substrate. Taken together, these observations indicate here for the first time that Cl- modulates Rb and cdc2, enhancing the proliferation of the MC3T3-E1 osteoblastic cells. (C) 2007 Elsevier Inc. All rights reserved.