The aim of the study was to examine the effect of morphine and naloxone pretreatment on cortisol and ACTH concentrations in response to machine milking in dairy cows. In the first part of the experiment, the effects of i.v. morphine doses (0, 21, 70 and 210 mg, one dose each day) 10 min before morning milking were studied in six Brown Swiss dairy cows. In the second part, four cows were treated 1 d after the control milking with 210 mg morphine at 10 min before milking and the next day with 210 mg naloxone at 15 min before milking followed by 210 mg morphine at 10 min before milking. In addition, four other cows were treated 1 d after the control milking with 210 mg naloxone at 10 min before milking. Pretreatment with morphine significantly suppressed the machine milking-induced increase of cortisol in blood plasma as compared with controls. Naloxone pretreatment overcame the inhibitory effect of morphine and elevated milking-induced cortisol concentrations. Naloxone administration alone significantly increased cortisol concentration resulting from milking as compared with controls. However, ACTH concentrations did not change in either control or treated animals, suggesting an ACTH-independent release mechanism for cortisol during milking. We conclude that the release of cortisol in response to machine milking seems to be modulated by endogenous opioids at the adrenal level and does not appear to be under the control of ACTH.