Serum and mammary tissue concentrations of progesterone and 20α-hydroxy-4-pregnen- 3-one (20α-OHP) were measured by competitive protein-binding assays and gas-liquid chromatography, respectively, in pregnant and lactating rats. The concentration of progesterone in mammary tissue of pregnant rats closely paralleled the serum concentration, particularly when tissue concentration was expressed as ng/mg DNA. The variability in tissue progesterone on the last day of pregnancy was relatively great, but there was a good inverse relation between the appearance of lactose and the progesterone concentration. Serum progesterone levels declined to their lowest values at 1–3 days of lactation (10 ± 1 ng/ml); the tissue concentration declined even more rapidly after parturition. The tissue 20α-OHP concentration, which was more closely related to serum progesterone among animals than to serum 20α-OHP, remained high after parturition suggesting that the presence of 20α-OHP has no effect on lactogenesis and that progesterone is decreased in the tissue by 20α-reduction. Following postpartum ovulation, serum progesterone increased to 74 ± 6 ng/ml at 6–9 days of lactation; tissue progesterone also increased to levels found in rats pregnant 14–19 days, yet no change in lactose content of the glands of suckled rats occurred, and the biosynthetic capacity in terms of the RNA/DNA ratio increased. Serum 20α-OHP also rose, but the tissue concentration was unchanged, suggesting that saturating levels were present throughout pregnancy and lactation. Since no rapid increase in DNA was associated with lactogenesis, differentiation of nonsecretory parenchyma] cells into daughter cells with the secretory capacity must occur earlier in pregnancy. Progesterone, therefore, must inhibit lactogenesis by preventing expression of the genetic potential of daughter cells. Once differentiation has been completed, however, the presence of progesterone in the tissue has no effect on the biosynthesis of milk constituents. (Endocrinology96: 861, 1975)