To test whether endogenous opioid peptides are involved in the behavioral and physiological responses of cattle to stress, 12 Holstein cows were either placed in social isolation in unfamiliar surroundings for 15 min or remained in their home stalls, either with or without naloxone treatment, following a Latin square design. Vocalizations (judged as high or low frequency), defecation/urination, and heart rate were recorded, latency to respond to local thermal stimulation of the leg by means of a laser was measured to detect pain sensitivity, and blood was sampled and assayed for cortisol concentrations. Naloxone in the home stall increased cortisol concentrations and tended to reduce response latencies to the laser but did not induce vocalization. Social isolation increased the incidence of high-frequency vocalization and of defecation/urination, heart rate, cortisol concentrations, and response latencies to the laser. Prior administration of naloxone increased the incidence of low-frequency vocalization in isolation, but it had no effect on heart rate or on responses to the laser and only limited effect on cortisol concentrations when the cows were isolated. Brief periods of social isolation in unfamiliar surroundings seem to be stressful to cows, as indicated by increased heart rate, hypothalamic-pituitary-adrenocortical axis activity, and vocalization. Isolation also reduces pain sensitivity, suggesting a stress-induced analgesia. However, we found no evidence that naloxone-sensitive opioid receptors were involved in these responses.