Human glandular kallikrein (hK2) is an androgen regulated protein primarily expressed in the prostate and recently identified as a novel prostate cancer marker. A 5 kb 5' flanking region of the hK2 gene was isolated and sequenced to characterize the regulatory mechanisms for the expression of hK2 in the androgen responsive prostate cell line, LNCaP. Using gene transfer, gel shift, and mutagenesis assays we have identified an ARE in the 5' far upstream promoter region of the hK2 gene that is crucial for its regulation in LNCaP cells. This study further demonstrated that the hK2 upstream ARE plays a predominant role in androgenic response. More interestingly, previously identified AREs in the prostate specific antigen promoter and the hK2 proximal promoter exert little activity in LNCaP cells. This study for the first time identifies a unique ARE that alone mediates the function of the androgen receptor in LNCaP cells in a cell dependent manner. This study also examines the activity of this ARE with 1alpha, 25 dihydroxy-vitamin D3 on the expression of the hK2 gene in LNCaP cells.