Objective: To investigate the expression of claudin-3 and claudin-4 in the eutopic and ectopic endometrium of women with endometriosis and to evaluate the role of claudin-3 and claudin-4 in the pathogenesis of endometriosis. Design: Cross-sectional measurement of gene expression levels of claudin-3 and claudin-4 on endometriotic tissue. Setting: Academic. Patient(s): Thirty-five patients with endometriosis and 35 healthy women who were free of endometriosis were recruited for the study. Intervention(s): Expression of claudin-3 and claudin-4 were investigated with immunohistochemical analysis, Western blot, and real-time polymerase chain reaction. Morphologic change of tight junction was also observed in different kinds of endometria. Main Outcome Measure(s): The expression levels of claudin-3 and claudin-4 in epithelial cells from 35 ectopic endometrial tissues, 27 eutopic endometrial tissues from women with endometriosis, and 35 normal endometrial tissues from women without endometriosis. Result(s): Expression of claudin-3 and claudin-4 was significantly lower in the ectopic endometriotic tissue than in the eutopic endometrium from women with endometriosis and normal controls at both the messenger RNA and protein levels. No significant difference was found between eutopic endometrium from women with endometriosis and normal endometrium from women without endometriosis. Conclusion(s): Down-regulated expression of claudin-3 and claudin-4 in ectopic endometrium suggests that claudin-3 and claudin-4 might play a pathogenic role in the formation of endometriosis. (Fertil Steril (R) 2009;91:1692-9. (C)2009 by American Society for Reproductive Medicine.)