The androgen-regulated 20-kDa protein gene consists of four exons that code for a major secretory protein of rat ventral prostate. Analysis of its potential cis-acting transcriptional regulatory elements revealed that a large intron 1 region (In-1) had stronger androgen response element (ARE) activity than did the 5'-flanking DNA. In cotransfected CV1 cells, In-1 and its most active subfragment In-1c functioned as AREs but not glucocorticoid response elements (GRE). Nevertheless several ARE/GRE-like partial palindromic sequences are present in In-1c, and it bound both androgen receptors and glucocorticoid receptors in mobility shift assays. A cluster of three ARE/GRE-like sequences contained within a 39-base pair sequence of In-1c had both ARE and GRE activities when analyzed as an isolated oligonucleotide, suggesting that other elements within In-1c determined its ARE specificity. In addition to ARE/GRE-like sequences, In-1c contains putative response elements for the transcription factors AP1, CREB, AP2, OCT-1, C/EBP, and a number of inverted and direct repeats. The ARE specificity of In-1c observed in CV1 cells was diminished in PC3 and HeLa cells transiently cotransfected with an androgen receptor or glucocorticoid receptor expression vector together with an In-1c reporter vector; however, the ARE activity of In-1c was greater than its GRE activity in these cell lines. Interestingly, a 131-base pair subfragment of In-1c retained ARE specificity in all three cell lines.