Previously, we reported that a small rat probasin (PB) promoter (-426 to +28 bp, -426PB) would target androgen regulated prostate-specific expression in transgenic mice. Later, we demonstrated that a Large (L) fragment (-10806 to +28 bp, LPB) of the PB promoter would target high levels of gene expression to the prostate in transgenic mice. These results suggested that optimal transcription of the PB gene depended on the presence of enhancer regions upstream of the proximal promoter. To identify these enhancers, the LPB fragment was sequenced and the enhancer activities of restriction fragments were characterized in cell lines. Two non-conventional androgen receptor binding sites (ARBS-3 and ARBS-4) in an upstream androgen-dependent enhancer of the PB gene were identified. One site functions as a weak steroid response element (SRE) in both LNCaP and MCF-7 cells; another site acts as a strong SRE, which preferentially responds to androgen and is preferentially activated in LNCaP cells. These two new ARBSs interact in a cooperative manner with the previously described Androgen Response Region (ARR defined by -244 to -96 bp) that contains ARBS-1, ARBS-2 and two lower affinity AR binding sites G-1 and G-2 sites. We conclude that the context in which the AR binding sites are present is pivotal in determining their effect on transcriptional regulation. Thus, the -705/+28 PB promoter contains a second androgen responsive region, probasin enhancer element (PBE, -705/-426 PB), in addition to the first described ARR. The PB promoter creates a model which contains six AR binding sites that function in a cooperative manner for maximum androgen-regulated prostate-specific gene transcription.