In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D 3 [1,25(OH) 2D 3] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH) 2D 3 traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH) 2D 3 called 1,25D 3-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D 3-MARRS expression modulates 1,25(OH) 2D 3 activity in breast cancer cells. Relative levels of 1,25D 3-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D 3-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH) 2D 3 in MCF-7 cells, a ribozyme construct designed to knock down 1,25D 3-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D 3-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH) 2D 3 ( IC 50 56 ± 24 nM) compared to controls (319 ± 181 nM; P < 0.05). Reduction in 1,25D 3-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH) 2D 3. Knockdown of 1,25D 3-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D 3-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH) 2D 3 in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D 3-MARRS expression or activity as anticancer agents.