Previously, an androgen responsive element (ARE or promoter ARE) was identified in the proximal promoter region of the prostate-specific antigen (PSA) gene. The proximal promoter fragment could mediate androgen induction of expression of a heterologous reporter gene in androgen receptor (AR)-less cells with exogenous AR in co-transfection assays. However, it exerted little androgen inducibility in androgen sensitive human prostate cells, LNCaP, which produce PSA mRNA and protein. In this study, we have identified a second functional ARE (or upstream ARE) approximately 4 kb upstream of the cap site of the PSA transcript. Interestingly, although the AREs are necessary for androgen induction, the DNA sequences surrounding the upstream ARE are also required for androgen induction by the PSA promoter in LNCaP cells. The results indicated that the upstream DNA sequences can cooperate with either ARE to mediate androgen induced gene expression in LNCaP cells.