Prostate cancer can be detected using assays for blood-borne prostate-specific antigen (PSA), which is the clinically most useful diagnostic marker of malignant disease. This paper characterizes the 5-flanking prostate-specific enhancer which controls expression of the human PSA gene This enhancer, located between ؊5824 and ؊3738, is androgen-responsive and requires a promoter for activity. Inductions of 12-100-fold activity occur at 1 nM concentrations of the testosterone analog R1881. The enhancer demonstrated tissue specificity as judged by transfections of several human cell lines. Electrophoretic mobility shift assays comparing nuclear extracts from breast cancer cells MCF-7, and prostate cancer cells LNCaP, showed three regions of prostatespecific binding. These three regions are ؊4168 to ؊4797 (region I), ؊4710 to 4479 (region II), and ؊4168 to ؊3801 (region III). Region III contained a putative androgen response element at ؊4136 that markedly affected activity if mutated. These data suggest that prostate-specific gene expression may involve interaction of prostatespecific proteins or protein complexes with the enhancer in addition to binding of the androgen receptor to androgen response elements.