We have established an in vitro system in which epithelial cells (EC) and stromal cells (SC) from the human endometrium are placed in culture to examine the production of CA125. There was a much greater amount of CA125 in heterotopic EC after culture cells reached confluence than during the logarithmic growth phase. Heterotopic EC in culture secreted nine times as much CA125 constitutively after reaching confluence as eutopic EC. Flow cytometry (FCM) analysis indicated that the changes in CA125 expression correlated with the cell cycle. The CA125 expression was mainly observed in the G0/G1-phase in the cell cycle. There was no amplification of the CA125 expression in heterotopic EC membranes. The results of SDS-PAGE and Western blot indicated that the 110 KDa molecule of CA125 might be specific for adenomyosis. The biochemical and physical nature of CA125 was examined to characterize the antigenic determinant of this antigen. These results strongly suggested that the CA125 antigenic determinant from EC was composed of conformationally dependent peptides. We conclude that significantly increased secretion of CA125 from heterotopic EC could be attributed to the increase in serum CA125 in patients with adenomyosis.