Aromatase, a cytochrome P-450, catalyzes the formation of aromatic C18 estrogenic steroids from C19 androgens. DNA sequence analysis of the human aromatase gene has revealed that a putative promoter sequence exists immediately up-stream of the second exon. Chloramphenicol acetyltransferase functional analyses of cells transfected with chloramphenicol acetyltransferase expression plasmids containing various DNA fragments derived from the 3'-end of the first intron of the aromatase gene were performed to show that a promoter indeed exists in this region. However, in all of the cell lines used in this study, MCF-7, JAR, OVCAR-3, and skin fibroblast, the function of this promoter was inhibited by a negative regulatory element situated up-stream from the promoter. The results further suggest that this inhibitory element behaves as a silencer element, in that it could inhibit a simian virus-40 promoter from a distance of several kilobases. This negative element worked in both orientations and inhibited the functions of several promoters, including the newly identified promoter situated in the 3'-end of the first intron of the human aromatase gene. Primer extension analysis has been performed to determine the potential transcription start site. The mechanism of the regulation of aromatase expression is known to be very complex. The presence of a promoter and a silencer at the 3'-end of the first intron may represent one additional way that aromatase expression is controlled in estrogen-producing cells.