To use dominant negative mutants of estrogen receptor genes delivered to endometriosis cells via an adenovirus vector (Ad-DN-ER) to abrogate estrogen action on these cells.Experimental in vitro study.University research laboratory.Patients with ovarian endometriomas provided endometriotic cells, and patients with uterine prolapse or subserous leiomyoma provided control endometrial cells.Transfection of endometriotic cells by dominant negative estrogen receptor genes via adenovirus vector (Ad-DN-ER).The main outcome measures were cellular proliferation, cytokine production, and induction of apoptosis in endometriotic cells.Coxsackievirus-adenovirus receptor mRNA expression and adenovirus transduction efficiency were significantly higher in endometriotic than normal endometrial cells. Ad-DN-ER–treated endometriotic cells, as compared with control virus–treated cells, showed cell rounding and detachment (cell death), a 72% reduction in the number of viable cells 5 days after transduction, significantly less production of monocyte chemotactic protein-1 (7.8 ± 0.5 vs. 152.8 ± 1.9 pg/mL, respectively), vascular endothelial growth factor (356.2 ± 11.6 vs. 997.3 ± 16.5 pg/mL, respectively), and interleukin-6 (268.7 ± 2.6 vs. 414.5 ± 3.6 pg/mL, respectively), and a significantly higher percentage of apoptotic cells (51.2 ± 7.8 vs. 23.8 ± 1.7, respectively).An adenovirus can effectively transfect endometriotic cells in vitro. The DN-ER delivered to endometriotic cells via an adenovirus decreases cell proliferation, induces apoptosis, and decreases cytokine production. Adenovirus-mediated gene therapy may represent a potential therapeutic option for endometriosis in the future.