Prednisolone is a steroid belonging to the corticosteroid group. The results obtained in the application of the 2008 and 2009 Italian Residue Control Plans show the frequent detection of prednisolone traces in cow's urine. Since most of the positive samples were detected at the slaughterhouse, the researchers hypothesised that, together with an increase of cortisol concentration, traces of prednisolone could be produced endogenously during stressful situations due to transport and handling before slaughter. In the present trial, 52 lactating cows housed in seven different farms in Lombardy, Italy, were studied. Urine samples were collected at the farm (after urethral catheterisation) and immediately after slaughter (from urinary bladder) together with 40 adrenal gland samples belonging to the same animals. All the samples were analysed for the determination of prednisolone and cortisol by LC/MS(n). The results demonstrated that prednisolone can be endogenously produced in dairy cows and, furthermore, its endogenous presence in bovine urine seems to be strongly related to a state of stress in the animals (at the farm and at the slaughterhouse). The data from adrenal glands do not, however, clarify if the endogenous production occurs, partially or totally, in this organ.