An earlier report in the literature indicated the vitamin D response element (VDRE) in the human parathyroid hormone (hPTH) promoter could be specifically bound by an unidentified transcription factor in addition to the vitamin D receptor (VDR) complex. We confirmed that OK and HeLa cell nuclear extracts formed a specific complex with the hPTH VDRE that was insensitive to competition with other VDRE sequences. However, this factor could be competed for by a consensus NF-Y DNA-binding site, and an anti-NF-Y antibody was able to supershift the bound band. Mutational analysis indicated that the NF-Y-binding site partially overlapped the 3' portion of the VDRE. Transfection studies using an hPTH promoter construct in Drosophila SL2 cells demonstrated strong synergistic transactivation by NF-Y interactions with both the VDRE site and a previously described distal NF-Y-binding site. Finally, mobility shift studies indicated that the VDR heterodimer competed with NF-Y for binding to the VDRE sequence, and NF-Y-stimulated activity of the hPTH promoter could be suppressed in a hormone-dependent manner when the VDR heterodimer complex was coexpressed in SL2 cells. In summary, these findings establish the presence of a proximal NF-Y-binding site in the hPTH promoter and highlight the potential for synergism between distal and proximal NF-Y DNA elements to strongly enhance transcription. Furthermore, findings suggest that the repressive effects of vitamin D on hPTH gene transcription may involve displacement of NF-Y binding to the proximal site by the VDR heterodimer, which subsequently attenuates synergistic transactivation.