The aromatase enzyme complex, located primarily in the stromal cells of breast tumors, catalyzes estrogen biosynthesis and is fundamental to hormone-dependent growth of breast cancer. Although an important pharmacological target, the mechanisms by which aromatase is regulated are poorly understood. Thus, regulation of aromatase activity and expression in human breast stromal cells by prostaglandin E(2) (PGE(2)) was investigated. PGE(2) exerts its actions via four transmembrane receptors, EP(1), EP(2), EP(3), and EP(4), which coordinate different signal transduction pathways. Using selective receptor agonists and antagonists, the involvement of the EP(1), EP(2), and EP(3) subtypes was assessed. Enzyme activity levels in cultures of disease-free stromal cells were determined using a tritiated water-release assay. PGE(2) and agonists of EP(1) and EP(2) significantly increased aromatase activity levels, which were decreased by the corresponding antagonists. An agonist of EP(3), an inhibitory pathway, antagonized activity levels induced by PGE(2). These results were generally reflective of changes in aromatase protein expression, determined by Western blotting analysis and the pattern of mRNA expression determined by a competitive RT-PCR method. Collectively, the results demonstrate that regulation of aromatase by PGE(2) is complex and may influence the development and progression of hormone-dependent breast cancer.