Prostate-specific-membrane-antigen (PSMA) is a marker protein expressed primarily in prostate epithelium. Its prostate-specific expression is conferred by PSMA enhancer (PSME), localized within the third intron of PSMA-encoding gene FOLH1. We recently reported that the 5'-end 90 bp of PSME harbored crucial enhancer elements for high PSMA expression. Deletion of this 90 bp sequence, called PSME(del3), significantly diminished PSME activity. We have further analyzed the regulatory elements in this 90 bp by transient transfection of linker scanning mutants. Two mutants, LN17 and 18, which harbored an AP-1 site and an AP-3 site, respectively, exhibited significantly lower enhancer activity. Subsequent site-directed mutagenesis changing the AP-3 site abolished the enhancer activity of PSME but not AP-1, indicating that AP-3 was the key cis-element enabling high PSMA expression. In addition, a 12 bp AP-3 site was able to enhance PSME(del3) activity by almost 40% higher compared to full-length PSME. However, AP-3 alone retained just the basal level of activity, indicating that the action by AP-3 was mediated by cooperation with other transcription factors binding to the PSME(del3) region. Transcription factor NFATc1 isoforms in nuclear extract were co-precipitated with the biotinylated AP-3 site by immobilized agarose beads and the genomic DNA containing PSME was precipitated by antibodies reactive to NFATc1, demonstrating that NFATc1 isoforms bound to the AP-3 site in PSME in vivo. Furthermore, ionomycin (calcium ionophore) and TPA augmented the enhancer activity of PSME, implying that calcium is an important regulator for PSMA expression in prostate cancer cell.