The aim of this study was to test if the opioid antagonist naloxone has a beneficial effect on normalization of oxytocin (OT) release during repeated milking of cows in unfamiliar surroundings. One control milking without naloxone treatment in all cows was performed in the familiar parlour. For four successive evening milkings, cows were transported to, and milked in, the operating theatre of the research station without (control group) or with naloxone administration (1 mg/kg BW) (naloxone group) before milking. After cessation of spontaneous milk flow, but not before 3 min of milking, vaginal stimulation was applied for 2 min. After milk flow ceased again, 10 IU of OT was injected intravenously to remove the remaining milk including residual milk. Milk flow was recorded continuously and blood samples were collected via a jugular vein cannula at 1-min intervals from 1 min before the start of milking until i.v. injection of OT. The inhibition of milk ejection and its normalization during repeated milking in unfamiliar surroundings was not influenced by naloxone treatment. Concentrations of cortisol and β-endorphin during control milking and all relocations were similar in the naloxone and control groups, although their concentrations were higher after relocations than in the control. Therefore, a role of endogenous opioids in the inhibition of milk ejection in unfamiliar surroundings could not be demonstrated. In addition, the effect of exogenous ACTH1–24 (8 IU, i.v.) on the release of cortisol related to the response of cows milked in unfamiliar surroundings was studied. Cows with totally inhibited milk ejection in response to vaginal stimulation during milking after first relocation had numerically, but not significantly lower cortisol levels (8·8±3·4 ng/ml; AUC/min) in response to ACTH than did cows with at least partial milk ejection (38·7±12·9 ng/ml). Thus animals with a higher adrenal response to ACTH seemed to have less severe inhibition of milk ejection.