In this study we examined the relationship between cortisol and inflammatory status in early lactating dairy cows after a stimulation test of the adrenal cortex. Twenty-four cows were grouped into quartiles (6 cows per each quartile) in accordance with the liver activity index (based on plasma concentration of negative acute phase proteins in early lactation); the quartiles were lower (LO; cows with the lowest liver functionality), intermediate lower, intermediate upper, and upper (UP; cows with the highest liver functionality). Each cow was injected i.v. with 20 μg of a synthetic analog of ACTH at 35 d in milk (DIM). Blood samples were taken to assess inflammatory status, and at 0, 30, and 60 min after ACTH challenge to measure total cortisol. The free cortisol fraction was analyzed in the LO and UP quartiles and the bound cortisol fraction was estimated as the difference between total and free cortisol. The LO, in comparison with the other quartiles, suffered a more severe inflammatory status, with the highest values of haptoglobin, reactive oxygen metabolites, and total nitric oxide metabolites and the lowest concentration of direct or indirect markers of negative acute phase proteins. The cows in the LO quartile had the highest values of plasma nonesterified fatty acids and β-hydroxybutyrate at 7 DIM, suggesting a more severe body lipid mobilization. The LO quartile cows showed the highest frequency of health problems and the lowest milk yield in the first 35 DIM. Thirty minutes after the ACTH treatment, the concentration of total cortisol was lower in LO in comparison to other groups. Similarly, the bound cortisol fraction was lower in LO versus UP. The adrenal response appeared inversely related with health status after calving (e.g., lower in LO cows, experiencing the most severe inflammatory status). The lower increase in cortisol after the ACTH challenge in cows with greater inflammation (LO quartile) seems a consequence of the lower availability of cortisol-binding globulin synthetized by the liver, but other mechanisms can be involved (e.g., rate of cortisol production, secretion, and metabolic clearance). Our data provide evidence that inflammation and metabolic changes reduce the concentration of circulating plasma cortisol during an acute stress. Hence, the acute phase response in dairy cows should be taken into account to interpret the results obtained from stimulation tests of the adrenal cortex. © 2013 American Dairy Science Association.