OK cells are a transformed cell line derived from opossum kidney proximal tubule cells. Prior studies have utilized this cell line to study both positive and negative transcriptional responses to Vitamin D. However, there was a noticeable decrease in sensitivity on the part of these cells to respond to Vitamin D treatment in transfection assays, particularly when assessing transcriptional activity from a heterologous promoter construct that used the chicken parathyroid hormone (cPTH) repressor Vitamin D response element (VDRE). Western blotting revealed the apparently diminished expression of both the Vitamin D receptor (VDR) together with its heterodimeric DNA-binding partner, the retinoid X receptor (RXR), in these cellular extracts. Co-transfection of either a VDR or RXR expression vector alone had little effect on hormone-dependent enhancer transcriptional activity from the human osteocalcin (hOC) reporter construct, or the degree of repression from the cPTH construct. Indeed, significant effects on repressor or enhancer activity were only observed in these cells when expression vectors for both the VDR and RXR were simultaneously introduced into the cells via transfection experiments. Analogous results were obtained irrespective of the identity of RXR isoform; co-transfection of either RXRα or RXRβ expression vectors together with the VDR-produced similar improvements in repressor activity. Titration of Vitamin D hormone under conditions of co-expression of the two receptors indicated that half-maximal responses were comparable for both VDREs and occurred at <1 nM concentration. In summary, these results are consistent with prior in vitro studies indicating interaction of the VDR with these VDREs occurs as a heterodimer complex with RXR. The decreased expression of both heterodimer partners observed in these cells could explain the requirement for additional VDR/RXR expression, in particular in order to compensate for the reportedly lower binding affinity of the heterodimer with the repressor cPTH VDRE. The extent of expression of both heterodimer partners, therefore, may act to modulate the available responses to Vitamin D in target cells.