Administration of 6IU synthetic ACTH1-24 intravenously to six Holstein-Friesian cows resulted in a cortisol peak concentration after 1 hour of 148 +/- 34.2 ng/ml. Basal plasma cortisol concentration (4.84 +/- 0.83 ng/ml) was reached 5 hours after ACTH injection. Until 7 days after ACTH administration no effect on milk yield was recorded. So it is concluded that a dose of 6 IU ACTH1-24 is sufficient for a conspicuous release of cortisol without any alteration in milk production. This dose can be used as a standard test for the evaluation of adrenocortical function in lactating cows when administered intravenously at 9 a.m. and when plasma samples for cortisol assay are collected just prior to administration and at 10 a.m.