Extraglandular estrogen synthesis mediates the proliferation of estrogen-responsive breast cancer in postmenopausal women. Aromatase, the cytochrome P450 Cyp19 enzyme, catalyzes the rate-limiting step in estrogen biosynthesis. Activity is present in both normal and neoplastic breast tissue, and Cyp19 protein is localized by immunohistochemistry predominantly in breast stromal fibroblasts. In cultured breast stromal fibroblasts, both activity and Cyp19 messenger ribonucleic acid are increased to a substantial degree by hormonal and growth factor regulators of transcription. Transcriptional regulation of CYP19 is complex in breast tissues, in which exon switching in the usage of alternative first exons occurs from predominantly EI.4 in breast tissue from cancer-free women to predominantly EI.3 and PII in breast tumors and quadrants with or without tumor. The present study questioned whether the first exon switch occurs as a result of an inherent difference between fibroblasts in normal and tumor tissues or because of differences in local regulators between these tissues. To distinguish between these two possibilities, we examined fibroblasts cultured from breast tumor, benign breast, and reduction mammoplasty tissues for the ability of various CYP19 transcriptional regulators to modulate first exon EI.3, EI.4, and PII usage. A semiquantitative RT-PCR method was used to identify transcripts containing six of the nine known CYP19 first exons. Combinations of cAMP and Dex regulated transcription from first exons EI.3, EI.4, and PII in fibroblasts cultured from all tissues, but not in reduction mammoplasty epithelial cells. These results provide evidence that the fibroblasts from these breast tissues are not inherently different in transcriptional regulation of CYP19 first exon usage and that transcriptional regulatory molecules are likely to mediate the exon switch phenomenon.