Aromatase, a cytochrome P450, catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogens. In this study, the regulatory properties of a 696-base pair region, that contains the promoter II and is situated immediately upstream of exon II of the human aromatase gene, were investigated. Chloramphenicol acetyltransferase (CAT) functional studies with DNA segments derived from this genomic region and primer-extension analysis revealed the presence of a second promoter which is functional in adipose stromal cells and in breast cancer cells. Detailed DNase-1 footprinting analysis, DNA mobility shift assays, and CAT functional studies of this genomic region were performed and led to the identification of a segment (B1) that could act as a promoter (probably promoter I.3) in adipose stromal and breast cancer cells. The study revealed further that the B1 region could be divided into two domains which were designated RE1 and RE2. RE1 was found to have the promoter activity, and RE2 was found to regulate the promoter activity of RE1, but in different manners in MCF-7 cells (as an example of breast cancer cells) and in adipose stromal cells. RE2 was found to function as a positive regulatory element in MCF-7 cells and as a negative regulatory element in adipose stromal cells, respectively. DNA mobility shift and UV-cross-linking experiments with BrUrd-substituted B1 fragment and nuclear extracts isolated from two types of cells were performed. The experiments identified DNA-bound proteins with molecular masses around 50 kDa. These findings serve as the basis for further examination of the regulatory mechanism of aromatase expression in human breast cancer and adipose stromal cells.