Steroidogenic factor 1 (SF-1) is a transcription factor shown to be critical for regulation of adrenal and gonadal development and function. To dissect the mechanisms that direct expression of this regulator, we have studied the promoter of the SF-1 gene and have identified cis-acting elements that recognize a basic-helix-loop-helix transcription factor; the CAAT binding factor; and Sp1. We demonstrate in Y1 adrenocortical cells that a 90-bp proximal promoter fragment is sufficient to direct steroidogenic-specific expression and that all three elements are required for activity of the SF-1 promoter. Functional analysis of the binding sites on a heterologous TATA box-containing promoter demonstrates that the CAAT box and Sp1 site are not essential for promoter activity when a TATA box is present, whereas the E box is absolutely required for gene expression and is most likely the steroidogenic cell-specific element. We also demonstrate that SF-1 itself does not significantly affect the transcription of its own gene, and thus conclude that the E box, CAAT box, and Sp1 site of the proximal promoter direct expression of the SF-1 gene.