Prostate specific membrane antigen (PSMA), also known as folate hydrolase (FOLH1), is a 100 kDa glycoprotein with elevated expression in prostate epithelial tissue. Expression of PSMA is upregulated as prostate tumor grade increases and is found in the vasculature of many tumors, with no presence in benign tissues. Due to the potential of the regulatory elements of the PSMA promoter and enhancer to be used in gene therapy and as biomarkers for prostate cancer under conditions of androgen ablation during treatment, we sequenced and analyzed the ability of 5.5 kb of PSMA promoter/leader region to promote transcription. A recently discovered enhancer, found in the third intron of the PSMA gene, FOLH1, was also studied. The promoter/leader region sequence provided basal expression in transcription assays, while addition of the enhancer activated transcription 41-fold in transient transfections and 144-fold in stable transfections of the LNCaP prostate cell line. This enhancement of transcription was not found in nonprostate cell lines or prostate cell lines that do not express PSMA. An analysis of the ability of androgens to act via the PSMA promoter/leader region and enhancer to activate transcription in transiently transfected LNCaP cells revealed no significant androgen response using the FOLH1 promoter/leader region and a downregulation of 42% with addition of the enhancer. In stably transfected LNCaP cells, the FOLH1 promoter/leader region produced a 21% downregulation in response to androgens, while addition of the enhancer resulted in a 45% downregulation. These results demonstrate significant upregulation of transcription by the PSMA promoter/enhancer, with specificity for the LNCaP prostate cell line, and downregulation of transcription in response to physiological levels of androgen.