The negative regulation of the human parathyroid hormone (PTH) gene by biologically active vitamin D-3 (1,25-dillydroxyvitainin D-3; 1,25(OH)(2)D-3) was studied in rat pituitary GH4Cl cells, which express factors needed for the negative regulation. We report here that NF-Y binds to sequences downstream of the site previously reported to bind the vitamin D receptor (VDR). Additional binding sites for NF-Y reside in the near vicinity and were shown to be important for full activity of the PTH gene promoter. VDR and NF-Y were shown to exhibit mutually exclusive binding to the VDRE region. According to our results, sequestration of binding partners for NF-Y by VDR also affects transcription through a NF-Y consensus binding element in GH4Cl but not in ROS17/2.8 cells. These results indicate that 1,25(OH)(2)D-3 may affect transcription of the human PTH gene both by competitive binding of VDR and NF-Y, and by modulating transcriptional activity of NF-Y. (C) 2005 Elsevier Inc. All rights reserved.