Background: Gene expression can be silenced through the methylation of specific sites in the promoter region. This mechanism of gene silencing has an important role in the carcinogenesis of prostate and other cancers. Although tumor-associated stromal cells also exhibit changes in gene expression, promoter methylation has not been described in these cells. Methods: Tumor epithelia, tumor-associated stroma and normal epithelia, and stroma adjacent to tumor tissues were isolated from whole-mount prostatectomy specimens (two per patient) of patients ( n = 5) with localized prostate cancer and from normal epithelia and stroma from benign prostate hyperplasia specimens (two per patient) from men ( n = 5) without prostate cancer by using laser capture microdissection or expression microdissection. The methylation status of three genes important in prostate carcinogenesis, GSTP1, RAR β2, and CD44, were evaluated using quantitative methylation-sensitive polymerase chain reaction. Results: GSTP1 and RARβ2 were methylated in the tumor epithelium of all five prostate cancer patients and in the tumor-associated stroma in four of the five patients. CD44 was methylated in the tumor epithelium from four of the five patients but not in the tumor stroma. GSTP1 and RARβ2 were methylated in normal epithelium of two and four patients, respectively, and in normal stroma of one and two patients, respectively, that were isolated from regions adjacent to the tumors and may have resulted from a tumor-field effect; CD44 methylation was not observed in normal epithelium or stroma. In contrast, normal epithelia and stroma from benign prostate hyperplasia specimens showed no promoter methylation in GSTP1, RARβ2, or CD44. Conclusions: The observation of promoter methylation in the non-neoplastic cells of the prostate tumor microenvironment may advance our understanding of prostate cancer development and progression and lead to new diagnostic and prognostic markers and therapeutic targets.