We tested the hypothesis that Mg 2þ influences growth of vascular smooth muscle cells (VSMCs) by modulating cell cycle activation through mitogen-activated protein (MAP) kinase-dependent pathways. Rat VSMCs were grown in culture medium containing normal Mg 2þ (1.02 mmol/L, control) and increasing concentrations of Mg 2þ (2-4 mmol/L) for 1-8 days. Effects of varying extracellular Mg 2þ concentration ([Mg 2þ ] e ) on intracellular free Mg 2þ concentration ([Mg 2þ ] i ) were assessed using mag-fura. Growth actions of Mg 2þ were evaluated by measuring cell cycle activation, DNA synthesis, and protein synthesis. Expression of cell cycle promoters, cyclin D1, cyclin E, Cdk2, and Cdk4 was assessed by immunoblotting. Phosphorylation of cell cycle inhibitors p21 cip1 and p27 kip1 and MAP kinases, ERK1/2, p38MAP kinase, and JNK was evaluated using phospho-specific antibodies. [Mg 2þ ] i increased in a dose-dependent manner in response to increasing [Mg 2þ ] e . These effects were evident within 2 days and maximal responses were obtained after 6 days. High [Mg 2þ ] e induced cell cycle activation with a lower proportion of cells in G 1 phase (75 AE 1.0%) and a higher fraction of cells in S phase (12 AE 0.7%) versus control (G 1 , 88.5 AE 1.4%; S, 6.8 AE 1.2%; P < 0.05). This was associated with increased protein content of cyclin D1 and Cdk4 and decreased activation of p21 cip1 and p27 kip1 . In cells exposed to 2 mmol/L Mg 2þ , DNA and protein synthesis was increased $threefold. Phosphorylation of MEK1/2 and ERK1/ 2 was enhanced two to threefold in cells grown in 2 mmol/L Mg 2þ . These effects were rapid, occurring within 2 days. Phosphorylation of MEK3/6, p38 MAP kinase, and JNK was unaltered by increasing [Mg2] e . PD98059 (10 À5 mol/L), specific MEK1/2 inhibitor, but not SB202190 (10 À5 mol/L) (specific p38 MAP kinase inhibitor), attenuated Mg 2þ -induced growth actions. These data demonstrate the novel findings that cell cycle activation and growth regulation by Mg 2þ occurs via ERK1/2-dependent, p38 MAP kinase-independent pathways.