The present study tested the hypotheses that 1) short-term (ST) dietary deficiency of magnesium (MgD; 21 days) in rats would result in the upregulation of neutral-, acid-, and alkaline- sphingomyelinases SMases) in cardiac and vascular smooth muscles (VSMCs), 2) ST MgD would result in an upregulation of proto-oncogenes, i.e., c-Fos and c-Jun, as well as the p65 and c-Rel components of NF-κB in cardiac and VSMCs, 3) low levels of Mg2+ added to drinking water would either prevent or greatly reduce the upregulation of the SMases and proto-oncogene expression, 4) exposure of primary cultured VSMCs to low extracellular Mg2+ concentration would lead to release of ceramide in both cerebral and aortic VSMCs, 5) specific inhibitors of neutral- and acid-SMAs would reduce the release of ceramide in cultured VSMCs exposed to low extracellular Mg2+, and 6) specific inhibitors of neutral- and acid-SMases would lead to reductions in the expression of c-fos, c-Jun, and NF-κB components. The data indicate that neutral-, acid-and alkaline-SMases exist in rat cardiac and VSMCs. ST MgD resulted in over 150% increases in SMase activity and proto-oncogene expression in left and right ventricular muscle, atrial muscle, and abdominal aortic smooth muscle; even very low levels of Mg2+ added to drinking water either prevented or ameliorated the activation of all 3-SMases as well as expression of c-Fos and c-Jun; scyphostatin and desipramine reduced the low Mg2+ - induced expression of the proto-oncogenes as well as p65 and c-Rel in VSMCs. Exposure of the VSMCs to low Mg2+ resulted in more than a 100% increase in release of ceramide; scyphostatin and desipramine reduced greatly the release of ceramide from the VSMCs. We believe when the present data are viewed in light of our previous, recent findings on the effects of Mg deficiency on most of the major enzymes in the sphingomyelin-ceramide pathway, that they could provide a rational basis for the treatment and prevention of drug-resistant hypertension, atherogenesis, and difficult-to-treat forms of cardiac failure.