Purpose: The metabolism and synthesis of intratumoral estrogens are thought to play a very important role in the etiology and progression of endometrial carcinoma. Aromatase is a key enzyme in the conversion of androgens to estrogens, and aromatase localization studies have reported that aromatase immunoreactivity and mRNA were detected mainly in stromal cells. However, the effect of tumor-stromal interactions on local estrogen biosynthesis in endometrial carcinomas remains largely unknown. Experimental Design: The endometrial carcinoma cell lines (Ishikawa and RL95-2) and breast carcinoma cell line (MCF-7) were cocultured with stromal cells isolated from endometrial carcinomas, and aromatization activity was measured using liquid chromatography-tandem mass spectrometry. We then confirmed the local biosynthesis of estrogens and tumor-stromal interactions on aromatase activity in Ishikawa and RL95-2 cells. In addition, we also examined the effects of aromatase inhibitors on cell proliferation. Results: Aromatase activity was significantly higher in cocultures with Ishikawa or RL95-2 than in each monoculture, respectively. Estrone (E 1 ) concentrations were significantly higher than estradiol (E 2 ) concentrations in Ishikawa and RL95-2 cells, whereas E 2 was significantly higher than E 1 in MCF-7 cells. Cell proliferation was significantly inhibited in Ishikawa and RL95-2 cell cultures treated with aromatase inhibitors compared with control cultures. Conclusions: These results indicate the contribution of not only E 2 but also E 1 to cancer cell proliferation in endometrial carcinoma. Our study may provide important information on metabolism and synthesis of intratumoral estrogens with regard to the etiology and progression of endometrial carcinoma, thus helping to achieve improved clinical responses in patients with endometrial carcinoma, who are treated with aromatase inhibitors. (Clin Cancer Res 2009;15(19):6028-34) Endometrial carcinoma is one of the most common malignancies in developed countries, and its incidence has increased (1). In situ estrogen metabolism, including synthesis and degradation, has recently been thought to play a very important role in the development and progression of various human estrogen-dependent neoplasms. The results of several studies have shown that the concentration of estradiol (E 2 ) in endometrial carcinoma tissue was significantly higher than the concentration in normal endometrium (2). Our results from a previous study were generally consistent with those of other investigations; we found that E 2 , estrone (E 1 ), and testosterone levels in tumor tissues were several times higher than concentrations measured in serum (3). These findings indicate that intratumoral estrogen metabolism and synthesis is important in the etiology and progression of endometrial carcinoma.Intratumoral production of estrogen occurs as a result of the aromatization of androgens such as androstenedione and testosterone into estrogens, and it is catalyzed by the cytochrome P450 aromatas...