The prostate-specific antigen (PSA) promoter (PSA-P) has been identified, characterized, and determined to be tissue specific. Compared with high expression of the genomic PSA gene in prostate cells, expression of the transgene driven by the putative PSA promoter is low. This suggests that the identified promoter may be incomplete or may function optimally with additional regulatory elements. To identify the presence of additional regulatory elements, we screened sequences upstream of the PSA promoter and identified a DNA fragment of 822 bp, which enhances PSA gene expression. Combining the newly identified PSA gene regulatory sequence (PSAR) with our previously identified PSA promoter (PCPSA-P) exhibited enhanced expressional activity in the PSA-producing LNCaP cell line. With the addition of 10 to 100 nM dihydrotestosterone, a more than 1000-fold increase in expression was observed as compared to androgen-negative controls. Furthermore, although the combined regulatory element (PSAR)-PSA promoter (PCPSA-P) sequence resulted in high transgene expression in LNCaP cell lines, the combined regulatory element-promoter sequence resulted in minimal expression in the non-PSA-producing prostate cell line PC-3, renal tumor cell line R11, and cervical adenocarcinoma cell line HeLa. The newly identified 822 bp alone could also function as a promoter. Compared with the combined promoter, however, the 822-bp fragment alone demonstrated lower activity and lower responsiveness to androgen stimulation. Our results suggest that coupling the PSA promoter with an upstream regulatory element results in a marked increase in PSA expression, suggesting that the complete PSA promoter contains two functional domains: a proximal promoter and a distal promoter, which can also function as an enhancer. The enhanced gene expression of the new construct, combined with its tissue specificity and androgen responsiveness, in turn provides a foundation for the development of tissue-specific vectors for prostate cancer gene therapy.