Eighteen cows had been selected for their responsiveness to psychological stress during the first lactation and were classified as having low (n = 10) or high (n = 8) cortisol concentrations in response to isolation-induced stress. In the present study these cows, now in their second lactation, were used to determine the effect of social isolation stress on the permeability of mammary tight junctions. During the experiment, each cow was isolated from the rest of the herd for 55 h. After the 1st h of isolation, each cow received a bolus infusion of endotoxin in one hind quarter in order to challenge tight junctions. Blood samples were taken throughout to measure lactose, which was used as an indicator of tight-junction leakiness. After 1 h of isolation, stress caused an increase in tight junction permeability in both groups, which was further enhanced by the endotoxin treatment. Although the permeability did not differ significantly between the two groups, it was consistently higher in the high-cortisol group, which was also the most stress-responsive group. Thus, psychological stress may adversely affect milk quality by allowing serum components to leak into milk.