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The concentration of progesterone in the milk of various species apparently has received only limited study (1, 2 ) . In the present project progesterone was assayed in the milk and blood plasma of cows during pregnancy.Materids and Methods. Blood and milk samples were collected from 56 Holstein-Friesian cows during their second or third pregnancy. Samples were collected from eight cows on each of the following days of pregnancy: 30, 60, 90, 120, 150, 180, and 210. Collections were made during the winter (November and December) and the cows were fed stored roughages (silage, hay) and grain consisting of 16% crude protein. The cows were not given any exogenous hormones or other drugs for several months prior to collection of samples.Milk (100 ml) and blood (20 ml) were collected approximately 2 hr after the morning milking. The blood was heparinized and both samples (milk and blood) were put immediately into an ice water bath and were stored in a refrigerator. The samples were transported to the laboratory every 3 or 4 days. The plasma was removed from the blood by centrifugation and the milk and plasma samples were stored in a freezer.Concentration of progesterone in the milk and plasma samples was measured by radioimmunoassay using a modification described by Abraham ( 3 ) . Radioactive progesterone ( 1 ,2-3H-progesterone, New England Nuclear) was purified on microcolumns of Sephadex . Approximately 2000 cpm of tritiated progesterone in 0.10 ml of a solution of 0.1% gelatin in phosphate buffer (0.1 % PBSG) were added to 4 ml of milk and 3 ml of plasma as an internal standard for recovery estimates. The milk samples were extracted by vortex mixing for 30 sec with 8-10 ml of diethyl ether followed by centrifugation at 2000 rpm for 10 min. The ether phase was removed with a disposable Pasteur pipet and evaporated to dryness under a stream of air in a water bath (38 C). This extraction procedure was repeated two more times. Plasma samples were extracted like the milk samples except that 5 ml of diethyl ether was used each time.Microcolumns of Sephadex LH-20 were prepared in 3-ml syringes which were plugged with glass fiber filters (grade 934 AH, Reeve Angel). Extracts from the plasma and milk samples were chromatographed on microcolumns containing 0.9 g of Sephadex LH-20 eluted with methylene dichloride: methanol (98: 2). The progesterone fractions ( 1-8 ml) were collected from the columns and were dried and rechromatographed.The Sephadex LH-20 (0.9 g) which was used for column chromatography of the progestins was slurried with 4-5 ml benzene: methanol (85 : 15). The columns were washed with 8-10 ml eluting solvent (hexane:benzene:methanol, 85 : 5:4) until all the air bubbles were removed. Another glass fiber filter was placed on top of the Sephadex and the columns were washed with an additional 10 ml of eluting solvent. The sample was applied to the column using two successive additions of 0.5 ml and the appropriate progesterone fractions (4-7 ml) were collected in scintilla- 354

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