Induction of tight junction permeability in the mammary epithelium decreases milk secretion, and in cows tight junctions become leaky after 17 h of milk accumulation. In vitro studies demonstrate the importance of glucocorticoids for the formation and maintenance of tight junctions. In this study we examined whether cortisol can prevent mammary tight junction permeability in the lactating gland in vivo, and inhibit the associated milk loss, using our milk-accumulation model to challenge tight junction patency. Following a 4-day control period Jersey cows were subjected to a 24-h period in which they were milked twice at 0700 and 1500 h (TM;n=6), once at 0700 h (OM;n=7), or once and treated with ACTH (40 IU per 2 h, starting after 14 h of milk accumulation) to increase endogenous cortisol levels (OM+ACTH;n=7). Frequent blood samples for cortisol, lactose and glucose analyses were taken via indwelling jugular catheters. ACTH treatment resulted in a sustained elevation of systemic cortisol concentrations. Plasma lactose, an indicator of tight junction leakiness, was not changed in TM cows, but began to increase rapidly at 17 h of milk accumulation in OM cows. Treatment with ACTH prevented the increase in plasma lactose, although levels were slightly, but not significantly, higher than in TM cows, indicating that elevated plasma cortisol reduced mammary tight junction leakiness. Milk yield was reduced by 12% in both once-milked groups, despite cortisol preventing tight junction leakiness. However, the milk loss in the latter group may not be related to leaky tight junctions, but be due to a reduction in milk precursor uptake by the mammary gland. Consistent with this notion was a 34% increase in plasma glucose levels in OM+ACTH cows only.