Prostate-specific antigen (PSA) is expressed primarily by both normal prostate epithelium and the vast majority of prostate cancers. Increases in serum PSA during endocrine therapy are generally considered as evidence for prostate cancer recurrence or progression to androgen independence. The mechanisms by which PSA up-regulation occurs in androgen-refractory prostate cancer cells are unknown. In this study, by using LNCaP and its lineage-derived androgen-independent PSA-producing subline, C4-2, we identified two cis-elements within the 5.8-kilobase pair PSA promoter that are essential for the androgen-independent activity of PSA promoter in prostate cancer cells. First, a previously reported 440-bp androgen-responsive element enhancer core (AREc) was found to be important for the high basal PSA promoter activity in C4-2 cells. Both mutation analysis and supershift experiments demonstrated that androgen receptor (AR) binds to the AREs within the AREc and activate the basal PSA promoter activity in C4-2 cells under androgen-deprived conditions. Second, a 150-bp pN/H region was demonstrated to be a strong AR-independent positive-regulatory element of the PSA promoter in both LNCaP and C4-2 cells. Through DNase I footprinting and linker scan mutagenesis, a 17-bp RI site was identified as the key cis-element within the pN/H region. Data from electrophoretic mobility shift analysis and UV cross-linking experiments further indicated that a 45-kDa (p45) cell-specific transcription factor associates with RI in prostate cancer cells and may be responsible for driving the PSA promoter activity independent of androgen and AR. Furthermore, by juxtaposing AREc and pN/H, we produced a chimeric PSA promoter (supra-PSA) that exhibits 2-3-fold higher activity than the wild type PSA promoter in both LNCaP and C4-2 cells.