An enzyme-linked immunosorbent assay (ELISA) was developed to measure anti-endometrial antibody concentrations in the serum of women with endometriosis. Pooled cytosolic protein extracts from the endometrial gland cells of 10 women were used as an antigen source. Serum samples were obtained from women with endometriosis before (n = 51) and after 6 months treatment with danazol or nafarelin (n = 30). Control sera came from women with a normal pelvis at laparoscopy, performed for sterilization (n = 23) or the investigation of pain and/or infertility (n = 22), 13 women with Rokitansky syndrome, and 10 umbilical cord bloods and adult males. There were no significant differences in serum anti-endometrial antibody concentrations before and after treatment, or between women with endometriosis and without endometriosis. Concentrations were lower in male and cord blood serum than in female's serum (P < 0.0001). We conclude that the ELISA is not a useful diagnostic tool for endometriosis unless more specific antigens can be isolated.