The mammalian ftz-f1 (mftz-f1) gene encoding Ad4BP/SF-1 has been demonstrated to be essential for the development of adrenal and gonadal glands. In a previous study, we identified an E box as the transcriptional element in the 5'-upstream region of the rat mftz-f1 gene. In the present study, we found a steroidogenic cell-specific transcriptional element in the first intron of the gene. Gel mobility shift and DNase I footprint analyses clearly revealed that Ad4BP itself binds to the element (Ad4 site). This finding was further supported by the positive effect of an Ad4BP expression vector on the transcription and by the significant decrease in the transcription caused by nucleotide substitutions within the Ad4 site. Similar loss was also caused by substitutions in the E box, indicating that the two elements are essential for the full transcriptional activity of the gene. DNase I hypersensitivity assays revealed that the chromatin structure around the Ad4 site and the E box was "open up" in the adrenal glands and Y-1 cells, whereas "closed down" in the liver. These observations indicated that the mftz-f1 gene is controlled by the autoregulatory loop in the steroidogenic tissues. The autoregulatory mechanism seems to be necessary to keep the mftz-f1 gene activated and thus maintain the tissues differentiated.