In a concerted analysis of the genes encoding three mouse steroid hydroxylases, we identified and characterized a transcriptional regulatory protein, designated steroidogenic factor 1 (SF-1), that contributes to the coordinate expression in adrenocortical cells. SF-1, an orphan member of the nuclear receptor family, binds to PyCAAGGPyCPu motifs upstream of the steroid hydroxylases to regulate their expression. In the present study, we extend these findings by examining the role of SF-1 in regulation of the rat P450 aromatase gene in gonadal tissues. The 5'-flanking region of the rat aromatase gene was isolated by a polymerase chain reaction-based approach, using primers corresponding to the 5'- and 3'-ends of a published aromatase sequence. DNA sequence analysis revealed three differences between our sequence and the previously published sequence, including a 44-base pair (bp) insertion. Moreover, the transcription initiation site, as determined by primer extension analysis, differed from that previously proposed. The new transcription initiation site is located 23 bp 3' of a putative TATA box. When a revised rat sequence was compared to that of the human aromatase PII promoter by BEST-FIT analysis, a region of about 300 bp was identified that was 80% conserved between the two promoters. A potential SF-1 site, CCAAGGTCA, was identified at position -82 within this region. An oligonucleotide probe containing this putative SF-1 site was used in gel mobility shift assays. Consistent with previous studies, a specific complex was observed with nuclear extracts from gonadal steroidogenic tissues but was absent with nuclear extracts from nonsteroidogenic tissues. The role of SF-1 in this steroidogenic cell-specific complex was next addressed more directly. Bacterial extracts containing an SF-1-glutathione S-transferase fusion protein interacted specifically with the putative SF-1 site, and polyclonal antisera against SF-1-glutathione S-transferase specifically abolished the complex formed with nuclear extracts from rat ovaries or R2C rat Leydig tumor cells. Finally, the aromatase SF-1 element increased expression of an SV40 promoter/luciferase construct in transient transfection experiments in a steroidogenic cell-selective manner. Collectively, these studies implicate SF-1 in the regulation of steroid hydroxylase gene expression in nonadrenal tissues, significantly extending previous studies in adrenocortical cells.