The androgen receptor (AR) is a ligand-dependent transcription factor that mediates androgen functions by regulating the expression of genes it targets. In the present study, we mapped 2 regions in human transforming growth factor β1 and cyclin-dependent kinase 2 promoters that negatively regulated transcriptional activity in an androgen-dependent manner in close proximity to positive androgen-response elements (pARE). We identified 7 negative androgen-response elements (nARE) in the negative androgen-response region in the TGF-β1 promoter. Each nARE is composed of 15 nucleotides and mediates androgen-dependent inhibition of transcription in close proximity of pAREs. The 5' portions of nAREs are highly conserved and resemble core half sites in pAREs. The nAREs interacted weakly with AR DNA-binding domain (AR DBD) in gel shift assays. Mutations on the conserved nucleotides in the nARE abolished its interaction with the AR DBD and abolished its inhibition of androgen-driven transcription, suggesting that nARE/AR interaction is essential for nARE-mediated inhibition of transcription. Taken together, these findings indicate that the AR negatively regulates the expression of its target genes via a negative androgen-response region composed of multiple nAREs.