Estrone (E1) and 17beta-estradiol (E2) are present in milk, but the mechanism(s) that regulate their appearance in milk are not known. The objectives of this study were to determine the impact of stage of pregnancy on the concentrations of E1 and E2 in plasma and milk and to determine the correlations between plasma and milk E1 and E2 and with milk components throughout pregnancy. Blood and milk samples were collected from 13 cows every 28 d throughout pregnancy. The E1 and E2 were quantified in plasma and milk using RIA after organic solvent extractions and Sephadex LH-20 column chromatography. Plasma E1 concentrations averaged 0.8, 16.9, and 41.8 pg/mL in trimesters 1, 2, and 3, respectively. The respective E1 concentrations in milk averaged 0.6, 7.9, and 27.1 pg/mL. The E2 concentrations in plasma averaged 0.5, 0.9, and 2.0 pg/mL; milk E2 averaged 0.3, 0.9, and 5.0 pg/mL. Plasma and milk E2 concentrations were greater in trimester 3 compared with trimesters 1 and 2. The E1 concentrations in milk were significantly correlated with plasma E1 concentrations (r = 0.77), percentage of milk fat (r = 0.50), and milk yield (r = -0.43). The E2 concentrations in milk were significantly correlated with plasma E2 concentrations (r = 0.93), percentage of milk protein (r = 0.63), and milk yield (r = -0.57). The milk-to-plasma ratio of E2 increased from 0.4 during trimester 1 to 2.2 in trimester 3, which suggested that the mechanism(s) regulating the appearance of E2 in milk may change over the course of pregnancy.