The hormone 1,25-dihydroxyvitamin D (1,25D) may play a protective role in prostate cancer. 25-hydroxyvitamin D 1-alpha hydroxylase (CYP27B1) is the enzyme responsible for the regulation of cellular 1,25D levels. CYP27B1 is substantially repressed in prostate cancer cells. We have investigated the molecular basis for this inhibition. First, we identify a repressive region between -997 and -1200 in the human CYP27B1 promoter following transient transfection analysis in the prostate cancer cell lines DU145, PC3 and LNCaP. Next, we demonstrate a role for the transcription factor growth factor independent-1 (GFI1) in the repression of CYP27B1. Electrophoretic mobility assays with nuclear extracts from prostate cancer cell lines established binding of GFI1 to the sequence 5'-TGGTACAATCATAACTCACTGCAG-3' present at -997 to -1200 in the repressive region. Site directed mutagenesis of the core GFI1 binding sequence (5'-AATC-3') substantially increased while forced expression of GFI1 decreased the expression of the CYP27B1 reporter construct. Importantly, GFI1 repression is dependent on an intact GFI1 binding site in the -997 to -1200 region. GFI1 is an oncoprotein known to form a large protein complex with co-repressors that recruit histone deacetylases. We propose that the formation of such a repressive complex on the inhibitory domain of the CYP27B1 gene in prostate cancer cells could lead to silencing of either the nearby enhancer or proximal promoter domains and lead to cancer progression by reducing local production of 1,25D. These studies provide the basis for a more detailed understanding of CYP27B1 repression in prostate cancer cells and could provide a novel insight in future diagnosis and treatment.