An in vitro, cell-free transcription system, based on prostate-derived transcriptional machinery and very powerful androgen response elements (AREs), has been developed. Multiple (p(ARR3)LovTATA) AREs from the androgen-regulated probasin gene were linked to G-free cassettes and used in nuclear extracts prepared from prostate carcinoma cell lines (PC3 and LNCaP cells) to test specific induction of transcription by full-length AR and by glutathione-S-transferase (GST)-fusion peptides in which the androgen receptor (AR) DNA-binding domain alone (AR524-649), or together with the ligand-binding domain (AR524-902), or a portion of the NH2-terminal domain (AR232-649) were incorporated. In the presence of AR, nuclear extracts from PC3 cells had greater activity in supporting transcription than those from LNCaP cells; and lower background activity than those from HeLa cells. All of the AR forms correctly initiated in vitro transcription of ARE-templates in an androgen-independent manner. The amount of specific, inducible transcript was dependent on the concentration of AR peptide present. AR524-902 was the most potent transactivator tested, with the maximal level of specific transcript over 900-fold higher than the minimal level. At all concentrations this peptide was three to four times more active than either AR524-649 or AR232-649. In conclusion, we have developed a very specific and sensitive cell-free transcription system for delineating trans-activational regions of the AR.