The present study tested the hypotheses that 1) short-term dietary deficiency of magnesium (21 days) in rats would result in the upregulation of sphingomyelin synthase (SMS) and p53 in cardiac and vascular (aortic) smooth muscles, 2) low levels of Mg 2+ added to drinking water would either prevent or greatly reduce the upregulation of both SMS and p53, 3) exposure of primary cultured vascular smooth muscle cells (VSMCs) to low extracellular Mg 2+ concentration ([Mg 2] o) would lead to the de novo synthesis of ceramide, 4) inhibition of either SMS or p53 in primary culture VSMCs exposed to low [Mg 2+] o would lead to reductions in the levels of de novo ceramide synthesis, and 5) inhibition of sphingomyelin palmitoyl-CoA transferase (SPT) or ceramide synthase (CS) in primary cultured VSMCs exposed to low [Mg 2+] o would lead to a reduction in the levels of de novo ceramide synthesis. The data indicated that short-term magnesium deficiency (10% normal dietary intake) resulted in the upregulation of SMS and p53 in both ventricular and aortic smooth muscles; even very low levels of water-borne Mg 2+ (e.g., 15 mg·l -1·day -1) either prevented or ameliorated the upregulation in SMS and p53. Our experiments also showed that VSMCs exposed to low [Mg 2+] o resulted in the de novo synthesis of ceramide; the lower the [Mg 2+] o, the greater the synthesis of ceramide. In addition, the data indicated that inhibition of either SMS, p53, SPT, or CS in VSMCs exposed to low [Mg 2+] o resulted in marked reductions in the de novo synthesis of ceramide. Copyright © 2010 the American Physiological Society.