In this study, the minimal promoter requirements of the TATA-less human androgen receptor (hAR) gene promoter are described. The hAR promoter is characterized by a short GC-box (-59/-32) and a long homopurine stretch (-117/-60). Two major transcription initiation sites, AR transcription initiation site I (AR-TIS I, (+1/2/3)) and AR transcription initiation site II (AR-TIS II, (+12/13)) are located in a 13-base pair region (Faber, P. W., van Rooij, H. C. J., van der Korput, J. A. G. M., Baarends, W. M., Brinkmann, A. O., Grootegoed, J. A., and Trapman, J. (1991) J. Biol. Chem. 266, 10743-10749). Transient transfection of COS cells with hAR promoter deletion and mutant constructs, followed by RNA isolation and S1 nuclease protection analysis showed that the process of transcription initiation through AR-TIS I and AR-TIS II is regulated by different promoter sequences. The GC-box directed initiation from AR-TIS II but did not affect AR-TIS I utilization, which is dependent upon sequences between positions -5 and +57. Band shift analysis identified the transcription factor Sp1 as the protein interacting with the GC-box. A single Sp1 binding sequence was found to be present in the GC-box. Footprint analysis confirmed the interaction of Sp1 with this sequence. The differential initiation through AR-TIS I and AR-TIS II was substantiated by the introduction of point mutations in the Sp1 binding sequence: only mutations that specifically abolished Sp1 binding interfered with AR-TIS II utilization, but all mutations left AR-TIS I initiation intact.