The steroid hormone 1&agr;,25-dihydroxyvitamin D3[1&agr;, 25-(OH)2D3] promotes vascular smooth muscle cell (VSMC) growth and calcification, but the precise mechanism by which 1&agr;, 25-(OH)2D3regulates VSMC migration is unknown. In rat aortic SMCs, we found that 1&agr;, 25-(OH)2D3(0.1 to 100 nmol/L) induced a dose-dependent increase in VSMC migration. This response required the activation of phosphatidylinositol 3-kinase (PI3 kinase) because 1&agr;, 25-(OH)2D3-induced migration was completely abolished by the PI3 kinase inhibitors, LY294002 (10 &mgr;mol/L) or wortmannin (30 nmol/L). Furthermore, the RNA polymerase inhibitor, 5,6-dichlorobenzimidazole riboside (50 &mgr;mol/L), did not affect 1&agr;, 25-(OH)2D3-induced VSMC migration, suggesting that gene transcription is not involved in this rapid response. Using analogs of 1&agr;, 25-(OH)2D3, which have been characterized for their abilities to induce either transcriptional or nontranscriptional responses of 1&agr;, 25-(OH)2D3, we found that 1&agr;,25-dihydroxylumisterol, which is a potent agonist of the rapid, nongenomic responses, was equipotent with 1&agr;, 25-(OH)2D3in inducing PI3 kinase activity and VSMC migration. Moreover, 1&bgr;, 25-(OH)2D3, which specifically antagonizes the nongenomic actions of 1&agr;, 25-(OH)2D3, abolished 1&agr;, 25-(OH)2D3-induced PI3 kinase activity and VSMC migration, whereas the inhibitor of the genomic actions of vitamin D, (23S)-25-dehydro-1&agr;-OH-D3-26,23-lactone, did not affect these responses. These results indicate that 1&agr;, 25-(OH)2D3induces VSMC migration independent of gene transcription via PI3 kinase pathway, and suggest a possible mechanism by which 1&agr;, 25-(OH)2D3may contribute to neointima formation in atherosclerosis and vascular remodeling.