Although transcription initiation within CYP19 (cytochrome P450 aromatase) occurs immediately 5' to the initiator methionine (proximal promoter) in two rat Leydig tumor cell lines (R2C and H540) that express high aromatase activity and in rat ovary, the patterns of aromatase expression in the two cell types are distinctive. To define mechanisms controlling different patterns of expression of the rat aromatase proximal promoter, we performed transient transfection and gel mobility shift assays. Transfection experiments using different sized promoter fragments fused to a reporter gene were used to identify regions that are functionally important for transcriptional regulation in steroidogenic cell lines [R2C, H540, and Y1 (mouse adrenocortical cells that express low aromatase activity)]. These experiments indicate that the cAMP response element (CRE) at –231 and the steroidogenic factor-1 (SF1) motif are both required for expression of the reporter gene in each steroidogenic cell line and that the CRE at –169 is similarly required in R2C cells. Gel mobility shift assays confirm binding of nuclear proteins from the steroidogenic cell lines to the SF1 motif and to CRE (–231). Leydig tumor cells also contain nuclear proteins that bind to the CRE (–169), but nuclear extracts from R2C cells produce a uniquely shifted band compared with H540 cells. These results suggest that differences in proteins that bind to distinct elements within the rat aromatase promoter may be responsible for different patterns and levels of aromatase expression in these steroidogenic cell lines. (Endocrinology 139: 5082–5093, 1998)