Mullerian Inhibiting Substance (MIS), a fetal testicular product that causes regression of the Mullerian duct in the male mammalian embryo, was evaluated for its antitumor effect on the premise that a substance active against this genital precursor in the fetus might also be active against tumors derived from these tissues. Increasingly pure fractions of biologically active MIS, prepared from newborn calf testes, were tested in the soft agar colony inhibition assay against single cell suspensions of fresh tumors derived in ascitic or solid form from patients with gynecologic malignancies. Twenty-eight tumor specimens placed in soft agar culture have provided sufficient growth to assess an MIS effect. Twenty-five of these 28 tumors showed significant colony inhibition after incubation with MIS. Increased antitumor response correlated with increased purification of MIS when the same tumor was treated with preparations of different purity. Samples obtained from the same patient at different times, from both ascites and solid tumor sources, produced nearly identical responses to MIS. MIS preparations, previously shown to be active in microcytotoxicity and colony inhibition assays against established human ovarian and endometrial carcinoma lines demonstrate consistent antitumor activity against fresh human gynecologic cancers removed at surgery.