Eight Holstein cows were utilized to examine the effect of prolactin on lactational performance prior to peak milk production (day 21-34 postpartum) and after peak milk production (day 60-73 postpartum). During each 14 day period, cows received daily intramuscular injections of pituitary-derived bovine prolactin (120 mg; 13.0 IU/mg protein) or excipient. Cows were housed in a controlled environment at 18.1C, 47.8% relative humidity and a 15 hr light: 9 hr dark cycle. In cows administered exogenous prolactin, circulating prolactin concentrations increased within one-half hr post injection, peaked within 2 to 6 hours and declined through the remainder of the day. Average prolactin concentration in the plasma was increased 2 to 5 fold over the 24 hr period in response to prolactin treatment. Yields of milk and milk components (fat, lactose and protein) were not affected by prolactin treatment in either period but the concentration of alpha-lactalbumin in milk was significantly increased (P less than .10) in both periods. Circulating concentrations of somatotropin, triiodothyronine, thyroxine, glucagon, nonesterified fatty acids and glucose were not altered. In prolactin-treated cows, the milking-stimulated prolactin release was decreased at both the PM milking, when circulating concentrations of prolactin were high, and the AM milking, when prolactin concentrations had returned to baseline. Concentration of prolactin in milk tended to increase but was not significantly altered by administration of exogenous prolactin. However, prolactin concentrations in plasma were correlated (r = .56) with milk concentrations. It is clear that postpartum administration of exogenous prolactin during the period of lactation prior to peak milk yield or after peak milk yield does not alter lactational performance in high producing dairy cows.