The androgen receptor (AR) mediates the biological functions of androgens and is essential for normal growth and differentiation of urogenital organs as well as initiation and maintenance of spermatogenesis. Withdrawal of androgens by castration or other methods has been shown to cause a marked, although often temporary, regression of many prostate cancers. In order to gain a better understanding of the transcriptional regulation of the AR, a series of truncation mutants derived from the 5′-region of the mouse AR (mAR) were inserted into the promoter-less plasmid pBLCAT3 and transiently expressed in the mouse α T3-1 and GT1-7 cell lines. The results of these experiments indicate the presence of a negative regulatory element in the 5′-untranslated region of the gene, which is able to reduce chloramphenicol acetyltransferase (CAT) activity by 77-89%. We have named this element the mAR suppressor (mARS). DNase-I protection assays of the 5′-untranslated region disclosed a protected domain. Gel mobility assays using the mARS revealed the presence of three protein-DNA complexes that could specifically bind to this protected domain. Insertion of the mARS into the thymidine kinase promoter containing pBLCAT2 vector resulted in a 2- to 10-fold decrease in CAT activity, but only if the insert was 3' to the start of transcription initiation. Finally, point mutations within the mARS were able to increase transcription of the AR promoter by 2.3-fold. The results of these experiments indicate that the mAR 5′-untranslated region contains a suppressor element. The suppressor is in a region that is bound by one or more proteins and is capable of suppressing the thymidine kinase promoter in a position-specific manner. Characterization of this suppressor may provide insight into the physiological means by which the AR is regulated in normal development and/or pathogenesis of androgen target tissues.