Pituitary transcription factor-1 (Pit-1) plays a key role in cell differentiation during organogenesis of the anterior pituitary, and as a transcriptional activator for the pituitary GH (GH) and prolactin (PRL) genes. However, Pit-1 is also expressed in non-pituitary cell types and tissues. In breast tumors, Pit-1 mRNA and protein levels are increased with respect to normal breast, and in MCF-7 human breast adenocarcinoma cells, Pit-1 increases GH secretion and cell proliferation. We report here that 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] administration to MCF-7 cells induces a significant decrease in Pit-1 mRNA and protein levels. By deletion analyses we mapped a region (located between -147 and -171 bp from the transcription start site of the Pit-1 gene) that is sufficient for the repressive response to 1,25-(OH)₂D₃. Gel mobility shift and chromatin immunoprecipitation (ChIP) assays confirmed the direct interaction between the vitamin D receptor (VDR) as homodimer (without the retinoid X receptor, RXR), and the Pit-1 promoter, supporting the view that Pit-1 is a direct transcriptional target of VDR. Our data also indicate that recruitment of HDAC1 is involved in this repressive effect. This ligand-dependent Pit-1 gene inhibition by VDR in the absence of the RXR seems to indicate a new mechanism of transcriptional repression by 1,25-(OH)₂D₃.