Synergisms and antagonisms between progesterone and other naturally occurring steroid hormones have been demonstrated by the application of the Hooker-Forbes bioassay for progestin activity (1-3). The assay is based on a particular biologic reaction, that of the stromal nuclei in the uterus of the ovariectomized mouse (4, 5 ) . The experiments to be reported were undertaken to determine the minimal effective dose (MED) of each of several steroid hormones when individually assayed and then to explore and quantify possible synergisms and antagonisms between progesterone and the other steroid hormones.Materials and methods. Separate stock solutions of progesterone, 16~-hydroxyprogesterone, 20~-hydroxyprogesterone, and 20P-hydroxyprogesterone were prepared by dissolving weighed amounts of each substance in ether, adding the quantity of sesame oil necessary to supply the desired concentration of the steroid, and removing the ether by distillation.The MED of progesterone and of 16~-hydroxyprogesterone in the CHI mouse are known (4, 6). Bioassays of these compounds and of 20a-hydroxyprogesterone and 20P-hydroxyprogesterone were carried out in young adult, ovariectomized B6D2F1 mice according to our usual procedure (4). All animals received food and water at will. The MED of a compound was determined by 1 This research was supported by Grant 5-ROI-HD-00456, National Institute of Child Health and Human Development, USPHS. Trivial names used in this article are progesterone = pregn-4-ene-3 ,2O-dione, 16a-hydroxyprogesterone = 16a-hydroxypregn-4en-3,20-dione, 20a-hydroxyprogesterone = 20a-hydroxypregn-4-en-3-one, and 20p-hydroxyprogesterone = 20ip-hydroxypregn-4-en-3-one.injecting each of a series of progressive dilutions into at least 5 uterine horns. The greatest dilution that would evoke a positive response of the stromal nuclei according to the criteria of the assay (elongated oval outline, conspicuous nucleolus, and fine, evenly scattered chromatin particles) in a majority of the horns in which it was placed was considered to represent the MED for that compound in that strain of mouse.Mixtures of two compounds were made by combining appropriate dilutions so as to give the desired proportions by weight of the steroids. I n the bioassay of such mixtures both the absolute and the relative amount of each hormonal component are important. A series of relative amounts, or proportions, was therefore selected, and a progression of absolute amounts at each proportion was assayed. As in the assay of a single compound, the end point for mixtures of progesterone and another steroid was regarded as the greatest dilution that would evoke a positive response in a majority of at least 5 uterine horns injected with that dilution. I n the early stages of an assay the absolute amount of progesterone injected into each horn was several times the MED of that hormone. Thus the ability, if any, of the second steroid to antagonize progestin activity was fully challenged. The assay of one mixture of two steroids in CHI mice is outlined in Tabl...