Tissue-specific aromatase production is significant in breast cancer and osteoporosis. Prostatic aromatase expression has been equivocal, and any local actions of estrogens are considered secondary to centrally mediated androgen suppression. We examine local aromatase expression and estrogen biosynthesis in the human prostate. Pure samples of stroma and epithelia from biopsy tissues were isolated by laser capture microdissection. Aromatase protein was detected by Western blot analysis, mRNA by RT-PCR, and enzyme activity by tritiated water assay, whereas promoter use was examined by real-time PCR. In nonmalignant prostate tissues, aromatase mRNA expression was absent from epithelium, but did localize to stroma. Presence of protein was confirmed, and expression was driven by promoter PII. Aromatase was expressed and active in LNCaP, PC3, and DU145 cells in addition to microdissected epithelial tumor cells; benign prostate epithelial cells showed no expression or activity. Promoter use in LNCaP and microdissected tumor cells was via PII, whereas PC3 and DU145 cells used promoter I.4. This study demonstrates local estrogen biosynthesis in prostate-induced aromatase gene expression in malignancy and potential alteration of aromatase promoter use with disease progression. These data provide a basis for continued investigation of local estrogen production and its potential role in prostate disease.