The epithelial cell adhesion molecule (Ep-CAM) exhibited an ovarian cancer:normal human ovarian surface epithelium ratio of 444. For validation studies, real-time quantitative PCR analysis and immunohistochemistry were performed in normal and malignant ovarian epithelial cell lines and tissues. To evaluate the potential of the Ep-CAM autoantibody as a tumor marker, we examined the amount of Ep-CAM autoantibody in serum samples obtained from ovarian cancer patients and normal controls by an ELISA. Real-time quantitative PCR analysis revealed significant overexpression of Ep-CAM mRNA in cancer cell lines (P < 0.001) and microdissected cancer tissues (P < 0.05), compared with that in cultured normal human ovarian surface epithelium and microdissected germinal epithelium, respectively. Immunolocalization of the Ep-CAM autoantibody showed that the sera of ovarian cancer patients expressed higher levels of Ep-CAM autoantibody than benign tumor patients and normal controls (P < 0.05). The levels of Ep-CAM autoantibody found were as follows: 0.132 in 52 patients with ovarian cancer, 0.098 in 26 cases with benign gynecologic disease, and 0.090 in 26 normal women. This investigation has shown that the Ep-CAM autoantibody was found to be associated with ovarian cancer and suggested that future research assessing its clinical usefulness would be worthwhile.