Apoptosis of vascular smooth muscle cells (VSMCs) plays an important role in remodeling of vessel walls, one of the major determinants of long-term blood pressure elevation and an independent risk factor for cardiovascular morbidity and mortality. Recently, we have found that apoptosis in cultured VSMCs can be inhibited by inversion of the intracellular [Na⁺]/[K⁺] ratio after the sustained blockage of the Na⁺,K⁺-ATPase by ouabain. To understand the mechanism of ouabain action, we analyzed subsets of hydrophilic and hydrophobic VSMC proteins from control and ouabain-treated cells by 2-dimensional electrophoresis. Ouabain treatment led to overexpression of numerous soluble and hydrophobic cellular proteins. Among proteins that showed the highest level of ouabain-induced expression, we identified mortalin (also known as GRP75 or PBP-74), a member of the heat shock protein 70 (HSP70) superfamily and a marker for cellular mortal and immortal phenotypes. Northern and Western blotting and immunocytochemistry all have confirmed that treatment of VSMCs with ouabain results in potent induction of mortalin expression. Transient transfection of cells with mortalin cDNA led to at least a 6-hour delay in the development of apoptosis after serum deprivation. The expression of tumor suppressor gene, p53, in mortalin-transfected cells was delayed to the same extent, and the expressed protein showed abnormal perinuclear distribution, suggesting that p53 is retained and inactivated by mortalin. Our studies therefore define a new [Na⁺]_i/[K⁺]_i-responsive signaling pathway that may play an important role in the regulation of programmed cell death in VSMCs.