The native PSA enhancer and promoter confer prostate-specific expression when inserted into adenovirus vectors capable of efficient in vivo gene delivery, although the transcriptional activity is low. By exploiting properties of the natural PSA control regions, we have improved the activity and specificity of the prostate-specific PSA enhancer for gene therapy and imaging applications. Previous studies have established that androgen receptor (AR) molecules bind cooperatively to AREs in the PSA enhancer core (-4326 to -3935) and act synergistically with AR bound to the proximal promoter to regulate transcriptional output. To exploit the synergistic nature of AR action we generated chimeric enhancer constructs by (1) insertion of four tandem copies of the proximal AREI element; (2) duplication of enhancer core; or (3) removal of intervening sequences (-3744 to -2855) between the enhancer and promoter. By comparing to the baseline construct, PSE, containing the PSA enhancer (-5322 to -2855) fused to the proximal promoter (-541 to +12), the three most efficacious chimeric constructs, PSE-BA (insertion of ARE4), PSE-BC (duplication of core) and PSE-BAC (insertion of core and ARE4), are 7.3-, 18.9-, and 9.4-fold higher, respectively. These chimeric PSA enhancer constructs are highly androgen inducible and retain a high degree of tissue discriminatory capability. Initial biochemical studies reveal that the augmented activity of the chimeric constructs in vivo correlates with their ability to recruit AR and critical co-activators in vitro. The enhanced activity, inducibility and specificity of the chimeric constructs are retained in an adenoviral vector (Ad-PSE-BC-luc). Systemic administration of Ad-PSE-BC-luc into SCID mice harboring the LAPC-9 human prostate cancer xenografts shows that this prostate specific vector retained tissue discriminatory capability compared with a comparable cytomegalovirus (CMV) promoter driven vector.