The concentrations of progesterone and 5alpha-pregnane-3,20-dione in ovarian and uterine venous plasma and in the systemic circulation were measured during gestation in the rat. The steroids were quantified by radioimmunoassay after separation on silicic acid micro-columns with the solvent system hexane : ethyl acetate (5 : 2, v/v). The concentration of progesterone in the systemic circulation was highest on days 3--4 and 13--17 of pregnancy; throughout gestation, the concentration of 5alpha-pregnane-3,20-dione was low in relation to that of progesterone and showed no marked changes as gestation proceeded. The level of progesterone in ovarian venous effluent was 10--20 times higher than that in the uterine vein and 20--50 times greater than that in the systemic circulation. The rate of secretion of progesterone by the ovary was highest during days 13--17 of gestation and ovariectomy during this period markedly reduced the levels of progesterone in the peripheral circulation. The concentration of progesterone in the uterine venous effluent was raised compared with the concentration in plasma from the abdominal aorta, especially on days 7 and 9 of pregnancy. These results suggest that, in vivo, the rat placenta synthesizes small amounts of progesterone and secretes it into the maternal circulation. The ovary is the primary source of progesterone during pregnancy and the placental contribution is of secondary importance. Although 4-ene-5alpha-reductase enzyme(s) is present in the ovary and placenta, significant quantities of the reduced progestin 5alpha-pregnane-3,20-dione are not secreted into the systemic circulation during gestation in the rat.