Cows were either exposed to 30 minutes (short) or 14 hours (long) of transportation. Cows that had experienced the long transportation were given a 24-hour rest period either by unloading them and allowing them to rest in a pen, or by allowing them to rest on the truck. Blood samples were taken before and after the short and long transportation and also after the rest period, which followed the long transportation. Short transport did not affect the secretion of adrenocorticotropin (ACTH) from lymphocytes. Long transport caused an increase in ACTH concentration. The ACTH concentration returned to the basal level in animals that were given the opportunity to rest for 24 hours in pens off the truck. Cows that were rested on the truck maintained elevated ACTH concentrations. Heart rates and rectal temperatures increased in cows that had experienced long transport, but decreased again after the 24-hour rest period (both on and off the truck). This study is the first to show lymphocytic ACTH secretion in cows. Based on the results from this study, adrenocorticotropin secretion from peripheral lymphocytes could be used as a reliable measurement in stress studies.