Ten Holstein cows, in second to fifth lactation, received 200 IU adrenocorticotrophin at approximately 4, 8, 16, 24, 32, and 40 wk of lactation. Blood samples were collected from indwelling jugular catheters at -60 and 0 min pre-injection and at 2, 6, 10, 15, 20, 30, 45, 60, 120, 180, 240, 360, 540, and 720 min postinjection. Post-injection concentrations of glucocorticoids were influenced by milk yield, stage of lactation, age, and mean environmental temperature but not by pre-injection concentration of glucocorticoids or days pregnant. As milk yield, cow age, and daily temperature (range 6.4 to 26.9 C) increased, glucocorticoid response decreased whereas it increased with advancing stage of lactation. Curves of adrenal response for the six injection times during lactation were not parallel, and average response adjusted for significant independent variables increased roughly in linear fashion from 15 to 53 ng/ml between 4 and 40 wk of lactation. Adrenal responsiveness was less in cows injected on days when temperatures were above 21.1 C than in those injected below this temperature. There was no evidence that adrenal function or milk production was altered in high producing cows at higher environmental temperatures differently from in low producing cows. The change in adrenal function during advancing lactation was not associated with pre-treatment peripheral plasma glucocorticoid concentrations which did not change. There appears to be a well mainained adrenal potential and sustained adrenocortical activity throughout lactation.