We investigated the action of 1␣,25-dihydroxyvitamin D 3 [1␣,25(OH) 2 D 3 ], a novel Gemini vitamin D 3 analog ], and a classic vitamin D 3 analog Ro-26-2198] in modulating the transforming growth factor- (TGF-)/bone morphogenetic protein (BMP) system in MCF10 immortalized breast epithelial cells. We found that 1␣,25(OH) 2 D 3 , Ro3582, and Ro2198 all enhanced BMP/Smad signaling by increasing the phosphorylation of receptor-regulated Smads. Ro3582 was more active than Ro2198, but both were considerably more active than 1␣,25(OH) 2 D 3. Ro3582 enhanced BMP/Smad signaling by 1) inducing the phosphorylation of receptor-regulated Smads (Smad1/5), 2) increasing the accumulation of phosphorylated Smad1/5 in the nucleus, and 3) activating BMPmediated transcription in MCF10 breast epithelial cells. Furthermore, Ro3582 induced the synthesis of BMP-2 and BMP-6 mRNA and protein, and the expression of Smad6 mRNA in MCF10 breast epithelial cells was inhibited by Ro3582. The induction of phospho-Smad1/5 by Ro3582 was inhibited by treatment with the BMP antagonist Noggin, whereas neutralizing antibody to TGF- did not block the induction of phosphoSmad1/5 by Ro3582. Treatment with Noggin also blocked the effect of Ro3582 on nuclear accumulation of phosphoSmad1/5 and the induction of BMP-2 and BMP-6 mRNA synthesis. These results indicate that the activation of BMP/Smad signaling by the Gemini vitamin D 3 analog Ro3582 may be through the production of BMP ligands, including BMP-2 and BMP-6, and/or down-regulation of the inhibitory Smad6. This is the first report to show that 1␣,25(OH) 2 D 3 and its derivatives activate BMP/Smad-specific signaling in human breast epithelial cells.The transforming growth factor- (TGF-) superfamily, including TGF-s, activins, and bone morphogenetic proteins (BMPs), are multifunctional cytokines that affect inflammation, immune responses, cell growth, differentiation, apoptosis, development, and carcinogenesis (Ten Dijke et al., 2002;Derynck and Zhang, 2003). BMPs are members of the TGF- superfamily regulating a large variety of biological responses in many different cells and tissues during embryonic development and postnatal life (Kawabata et al., 1998;Miyazono et al., 2005). BMPs exert their biological effects via binding to two types of serine/threonine kinase BMP receptors, activation of which leads to phosphorylation and translocation into the nucleus of intracellular signaling molecules, including Smad1, Smad5, and Smad8 (Kawabata et al., 1998). Upon BMP receptor activation, the BMP receptor-regulated Smads are phosphorylated in the C-terminal of the MH2 domain and recruit the common partner Smad, Smad4, to the nucleus to mediate BMP-dependent target gene expression (Kawabata et al., 1998;Miyazawa et al., 2002;Miyazono et al., 2005).Although BMPs belong to the TGF- family and mainly