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A short-term treatment with estradiol-17 beta and progesterone induced mammary gland growth and lactogenesis in non-pregnant ewes. These events normally occur during the second half of pregnancy. In order to compare the hormonal environment during the treatment to that during pregnancy, the concentrations of total estrogens (TE), progesterone (P4), glucocorticoids (G) and prolactin (Prl) in the plasma were characterized in non-pregnant, intact ewes induced into lactation with subcutaneous injections of estradiol-17 beta (E2-beta) and P4 for 7 consecutive days (days 1 to 7). Eight non-pregnant, intact, multiparous ewes were divided into two groups (groups I and II, 4 ewes each), according to their milk yields recorded during the previous lactation. All the ewes received the short-term treatment with E2-beta and P4; the ewes of group II were also injected with hydrocortisone acetate (H) and growth hormone (GH) twice daily on days 18 to 20. Blood was collected twice daily for 21 days and milking was initiated 19 days after the first injection of E2-beta and P4. Concentrations of TE, P4, G and Prl were measured in the plasma of ewes in group I but only G and Prl were measured in the plasma of the ewes in group II. Two ewes of group I and one of group II failed to lactate. Mean milk yields (2.5 +/- 0.4 and 6.3 +/- 0.9 kg/34 days) were highest when injections of H and GH were included. Average pretreatment concentrations of plasma TE and P4 were 3.1 +/- 0.8 and 2.1 +/- 0.4 ng/ml, respectively; these increased to 10.2 +/- 1.3 and 5.6 +/- 0.6 ng/ml during the week of the E2-beta and P4 injections. Peak concentrations occurred on days 7 and 6, respectively, and the plasma levels had returned to pretreatment values by days 18 to 20. Concentrations of G in plasma were higher (13.0 +/- 1.0 ng/ml) during E2-beta and P4 injections than during pre- or post-injection (7.4 +/- 1.4 and 8.2 +/- 0.7 ng/ml, respectively). Plasma Prl showed biphasic two to six-fold increases on days 4 and 7 during E2-beta and P4 injections and thereafter declined to lower average daily concentrations on days 10 to 13. Plasma Prl began to increase, and its average concentration was 579 +/- 56 ng/ml by days 18 to 20. These results suggest that the amount of the P4 injections should be increased and that the treatment with E2-beta and P4 should last long enough to insure the induction of an endocrine balance which would correspond more accurately to the hormonal events occurring during the second half of pregnancy in the ewe.