In this study, we used a 5'-flanking region (-426/+28) of the rat prostatic probasin (rPB) gene shown to be sufficient to direct prostate-specific expression in transgenic mice in identifying the exact DNA-binding site of a putative prostate-specific transcription factor. Chloramphenicol acetyl transferase (CAT)-reporter gene analyses revealed that the construct pCAT PB -244/+52 was equally well induced by androgens in both prostatic LNCaP and nonprostatic COS-1, MCF-7, HEC-1, and HEP-1 cell lines, indicating that although the probasin gene region -244/+52 was important for androgen regulation, it was not regulated in a prostate-specific manner. Further studies suggested that the region -278/-240 was most crucial for prostate-specific expression. The sequence -426/-279 could be considered a silencer area, especially in nonprostatic cells. In deoxyribonuclease I footprinting, a protected 12-bp region was found between the nucleotides -251 and -240 only with nuclear extracts of prostatic origin. Deletion of this area decreased androgen induction significantly (P < 0.05) in transient transfections of prostatic cells compared with the wild-type reporter construct. Glucocorticoids were incapable of increasing the induction of the pCAT PB -278/+52 reporter construct compared with that of pCAT PB -244/+52 in the prostatic cell line LNCaP, suggesting that the putative prostate-specific protein acts as an inducer only when androgen and androgen receptor are present.