Vitamin D interacting protein 205 (DRIP205) is a mediator complex protein that anchors the complex to the estrogen receptor (ER) and other nuclear receptors (NRs). In ZR-75 breast cancer cells treated with 17?-estradiol (E2) and transfected with a construct containing three tandem estrogen responsive elements (pERE3), DRIP205 coactivates ER?-mediated transactivation. DRIP205?587-636 is a DRIP205 mutant in which both NR boxes within amino acids 587-636 have been deleted and, in parallel transfection studies, DRIP205?587-636 also coactivates ER?. Moreover, both wild-type and variant DRIP205 also colocalize with ER? in the nuclei of transfected cells. AF1 and AF2 of ER? are both required for DRIP205 coactivation. Extensive deletion analysis of DRIP205 shows that multiple domains of this protein play a role in coactivation of ER? and in interactions with ER?. On the other hand, both DRIP205 and DRIP205?587-636 coactivate E2-induced transactivation of ER?/Sp1 in cells transfected with a construct containing three GC-rich sites (pSp13). Coactivation of ER?/Sp1 by DRIP205 is dependent on AF1 of ER?. Enhancement of ER? and ER?/Sp1 by DRIP205 does not require NR boxes of DRIP205, and deletion mutants DRIP205 (1-714) and DRIP205 (516-1566) significantly coactivate ER? and ER?/Sp1. RNA interference study showed that DRIP205 coactivation of ER?/Sp was abolished in cells transfected with iSp3 and iSp4, suggesting that Sp3 and Sp4 are required for coactivation of ER?/Sp by DRIP205 in ZR-75 cells.