The functional and structural interactions of two androgen receptor-binding sites in the 5'-flanking DNA of the rat probasin gene were determined. Deletion mapping and DNase I footprinting analysis had previously identified two androgen receptor-binding sites (ARBS) necessary for androgen induction of the probasin gene: ARBS-1, which resembled a glucocorticoid-responsive element, and ARBS-2, which had a unique sequence. In this study, maximal androgen induction in transient transfection studies only occurred when both sites were present. Neither binding site functioned independently, and deletion of the DNA sequence between the sites resulted in a 60% loss of androgen inducibility. Moreover, point mutations in either ARBS-1 or ARBS-2 led to > 90% loss in activity. Scatchard analysis indicated that ARBS-1 and ARBS-2 bound a synthetic androgen receptor, AR2, with Kd values of 20.0 and 6.7 nM, respectively. Consistent with the higher affinity, ARBS-2 bound AR2 at half the threshold concentration (200 ng) of that required in reciprocal DNase I footprinting experiments with ARBS-1. By comparison, protection occurred at a much lower threshold concentration of AR2 (60 ng) and to the same extent over each site when both sites were present, suggesting a cooperative interaction between the two sites. The cooperative effect was further substantiated when a point mutation in ARBS-1 blocked AR2 binding not only to ARBS-1, but also to ARBS-2. Similarly, a point mutation in ARBS-2 also prevented receptor binding to both sites. Androgen-specific regulation of probasin gene transcription therefore required an androgen-responsive region (positions -286 and +28) containing two androgen receptor-binding sites, where the binding of the androgen receptor to both sites occurred in a cooperative, mutually dependent manner.