Vascular calcification is a common finding in atherosclerosis and a serious problem in uremic patients. Because of the correlation of hyperphosphatemia and vascular calcification, the ability of extracellular inorganic phosphate levels to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro was examined. HSMC cultured in media containing normal physiologic levels of inorganic phosphate (1.4 mM) did not mineralize. In contrast, HSMC cultured in media containing phosphate levels comparable with those seen in hyperphosphatemic individuals (>1.4 mM) showed dose-dependent increases in mineral deposition. Mechanistic studies showed that elevated phosphate treatment of HSMC also enhanced the expression of the osteoblastic differentiation markers osteocalcin and osf2/Cbfa-1. The effects of elevated phosphate on HSMC were mediated by a sodium-dependent phosphate cotransporter (NPC) as indicated by the ability of the specific NPC inhibitor phosphonoformic acid to dose-dependently inhibit phosphate-induced calcium deposition as well as osteocalcin and Cbfa-1 gene expression. The NPC in HSMC was identified as Pit-1, a member of the novel type III NPCs. These data suggest that elevated phosphate may directly stimulate HSMC to undergo phenotypic changes that predispose to calcification and offers a novel explanation of the phenomenon of vascular calcification under hyperphosphatemic conditions. Furthermore, we examined the factors affecting peripheral vascular calcification in 332 nondiabetic hemodialysis patients. There were 45 nondiabetic patients with vascular calcification. In multivariate logistic regression, the significant factors affecting vascular calcification were advanced age, longer duration of hemodialysis, increased phosphate concentrations, male gender, and lower predialysis diastolic pressure. Our findings suggest that an elevated phosphate level may directly stimulate HSMC to undergo phenotypic changes that predispose to calcification and offer a novel explanation of the phenomenon of vascular calcification under hyperphosphatemic conditions.