The expression of P450 aromatase and other steroidogenic enzymes were evaluated in 42 endometrioid endometrial carcinomas, 23 endometrial hyperplasias, and 7 normal endometrial specimens. These findings were correlated with clinicopathological findings to elucidate the possible biological significance of in situ estrogen production in the development of human endometrial carcinoma. Only weak aromatase immunoreactivity was observed in vascular walls and myometrial cells. In contrast, strong aromatase stromal immunoreactivity was observed in 28 of 42 (66.7%) endometrial carcinomas. However, no stromal immunoreactivity was seen in normal or hyperplastic endometrial specimens. Immunoreactivity in the carcinoma stromal cells was significantly increased at sites of invasion. These aromatase-positive cells were immunohistochemically negative for other steroidogenic enzymes involved in estrogen biosynthesis. In situ hybridization studies revealed aromatase mRNA hybridization signals in stromal cells but not in carcinoma cells. The distribution of aromatase mRNA correlated well with the immunohistochemical localization of aromatase enzyme. Quantitation of aromatase activity demonstrated 8.75 +/- 2.75 pmol/hour/mg of protein for endometrial carcinomas (22 specimens) and 0.98 +/- 1.95 pmol/hour/mg of protein for normal endometrial specimens (4 specimens). Aromatase activity was found in both estrogen receptor-positive and -negative endometrial carcinomas. Aromatase did not vary with respect to the menopausal status of patients with endometrial carcinoma. These results suggest that estrogen is produced in situ in endometrial carcinoma but not in benign endometrial lesions. Such locally synthesized estrogen may act on carcinoma cells in a paracrine fashion to promote tumor growth. Additional investigations are necessary, but increased aromatase expression in the stromal cells of endometrial carcinoma may therefore play an important role in the development of human endometrioid endometrial carcinoma.