Several studies demonstrate an association between osteoporosis and arterial calcific disease both of which being common in elderly women. Estradiol and raloxifene, a selective estrogen receptor modulator, prevent bone loss in post-menopausal women. Little is known regarding how these agents affect arterial calcification. The aim of this study was to determine whether or not 17-estradiol and raloxifene reduced vascular smooth muscle cell differentiation and expression of bone-associated proteins during phosphate-induced calcification in vitro. Aortic smooth muscle cells (VSMC) were cultured from adult gonadally intact and ovariectomized (OVX) female pigs. Calcifying media was added and cells were treated with solvent (control), 17-estradiol (E₂) or raloxifene. Extent of calcification and phenotypic expression of bone-associated proteins [matrix gla protein (MGP), osteoprotegerin (OPG) and bone sialoprotein (BSP)] were examined at three-day intervals over two weeks. Calcium content increased in all groups but was greater in VSMC derived from intact compared to OVX animals. E₂ reduced calcification and preserved a contractile phenotype. Expression of OPG significantly decreased with time; this decrease was significantly greater in VSMC derived from OVX compared to gonadally intact pigs. E₂ and raloxifene preserved expression of OPG only in VSMC from intact pigs. Expression of MGP increased significantly with time and was not affected by E₂ or raloxifene treatments. E₂ treatment significantly inhibited synthesis of BSP in cells from both groups. In conclusion, 17-estradiol slows differentiation of VSMCs induced by excess phosphate. Effectiveness of raloxifene to preserve expression of bone cell-associated proteins depends upon the hormonal status of the tissue donor.