Müllerian inhibiting substance (MIS) is a fetal testicular product which causes regression of the müllerian duct in the male mammalian embryo. This material has been partially purified from a neonatal bovine source and in cruder fractions has shown antitumor effects when tested against the HOC-21 ovarian carcinoma line in monolayer cytotoxicity, in soft agar colony inhibition assay, and in nude mouse xenografts. The glycoprotein used for the present studies was more highly purified by sequential ion exchange, carbohydrate affinity, and dye affinity chromatography. After a 1-hr exposure with 1.0 × 10 6 tumor cells prior to heterotransplantation, this more purified preparation with MIS biological activity as determined by organ culture assay of embryonic urogenital ridges delayed the appearance of palpable tumor nodules. That this response may be specific for tumors derived from the coelomic epithelium of the embryo is further supported by the absence of any antitumor effect when this substance was tested against the SW-48 colon carcinoma line. It is of interest that the antitumor activity followed the biological activity as the preparation was further purified to 30,000-fold.