The aim of the study was to determine the role of opioids modulating the release of prolactin (PRL) in response to milking in sixteen Brown-Swiss dairy cows. Two experiments were carried out to measure the dose-related effect of morphine and the effect of the opioid antagonist naloxone (NAL), with or without morphine. In the first experiment, six cows were injected (via catheter) on 3 consecutive days after the control milking (0 mg) with 21, 70 and 210 mg morphine-HCl 10 min before the start of the morning milking. The second experiment was divided into two parts. In the first part, four cows were injected after control morning milking with 210 mg morphine, 10 min before the start of the following morning milking. This was followed on the next day by an application of 210 mg NAL (15 min before the start of milking) and 210 mg morphine. In the second part, after control milking for 1 d, six cows were injected with 210 mg NAL 10 min before milking. Morphine at the highest dose tended to stimulate basal PRL levels in the first and second experiments (P<0·10). PRL increased in response to machine milking but morphine did not stimulate its release further. NAL alone, or when given with morphine did not influence the release of PRL in response to machine milking. However, NAL was effective in suppressing the increase in basal levels of PRL caused by morphine. In conclusion, although morphine tended to stimulate basal levels of PRL before milking, the release of PRL during milking seemed not to be regulated by opioids.