An experiment was conducted to determine the effect of iv administration of adrenocorticotropin (ACTH) and(or) prolactin on serum cortisol and progesterone concentrations in hypophysectomized, ovariectomized, adult ewes. Twenty-four hours after surgery (d 1), six sheep were used in a crossover design and were assigned randomly to receive iv one of four treatments: (I) 200 IU ACTH in 10 ml .15 M NaCl at 0700 h, (II) 2.5 mg prolactin in 10 ml .15 M NaCl at 0700 h and again at 0730 h, (III) 200 IU ACTH and 2.5 mg prolactin in 10 ml .15 M NaCl at 0700 h followed by 2.5 mg prolactin in 10 ml .15 M NaCl at 0730 h, or (IV) 10 ml .15 M NaCl (vehicle) at 0700 and 0730 h. Blood samples (5 ml) were collected via jugular cannulae at 15-min intervals from 0600 to 0700 h and starting at 0700 h at 5, 10, 15, 30, 35, 40, 45, 60, 75, 90 and 120 min. Samples were then collected at hourly intervals until 1300 h. Twenty-four hours after commencement of first sampling (d 2), treatments were crossed and samples were collected as on d 1. Administration of ACTH alone or in combination with prolactin increased (P less than .01) serum cortisol and progesterone concentrations. Serum cortisol or progesterone concentrations were unaffected (P greater than .05) by administration of prolactin.