Ad 4 BP, also known as SF-1, is a steroidogenic tissue-specific transcription factor that is also essential for adrenal and gonadal development. Two mechanisms for the transcriptional regulation of the mammalian FTZ-F 1 gene encoding Ad 4 BP in adrenocortical cells have been proposed in the previous studies: the crucial role of a cis-element, an E box for the steroidogenic cell-specific expression of mouse and rat FTZ-F 1 genes, and a possible autoregulatory mechanism of the rFTZ-F 1 gene by Ad 4 BP itself through binding to the Ad 4 (or SF-1) site in the first intron. In the present study, the transcriptional regulation of the human FTZ-F 1 gene in adrenocortical cells was investigated from several angles, including the above two mechanisms. Using a series of deletion analyses of the 5'-flanking region of the hFTZ-F 1 gene and site-directed mutagenesis for transient transfection studies, an E box element, CACGTG at -87/-82 from the transcriptional start site, was also found to be essential for the transcription of the hFTZ-F 1 gene in mouse or human adrenocortical cell lines as well as in non-steroidogenic CV-1 cells. Despite the presence of a corresponding Ad 4 site, CCAAGGCC at +163/+156 in the first intron of the hFTZ-F 1 gene, an autoregulatory mechanism through the Ad 4 site was found to be unlikely in the hFTZ-F 1 gene mainly due to site-directed mutagenesis. In addition, the forced expression of Ad 4 BP had little effect on hFTZ-F 1 gene transcription in non-steroidogenic CV-1 cells. Such Ad 4 BP-independent regulation of the hFTZ-F 1 gene was in striking contrast to the regulation of steroidogenic CYP genes, such as the human CYP 11 A gene, in which the proximal promoter activity is Ad 4 BP-dependent and the transactivation by Ad 4 BP is silenced by DAX-1. Even though the Ad 4 BP-dependent transcriptional regulation of the DAX-1 gene has been reported, DAX-1 did not affect the transcriptional activity of the hFTZ-F 1 gene in our study. Taken together, these observations suggest that the E box is indeed required for the expression of the FTZ-F 1 gene, at least in mammalian species, but may not determine the tissue-specific expression of the hFTZ-F 1 gene, and that, unlike the steroidogenic CYP gene, the regulation of the hFTZ-F 1 gene appears to be independent of both Ad 4 BP and DAX-1.