Abstract After a two-year period of the frequent detection of prednisolone-positive bovine urine samples in the Italian region of Lombardy, studies were initiated to investigate the source. Because the majority of positive samples were detected at the slaughterhouse, researchers hypothesised that, together with increased cortisol and cortisone, a small quantity of prednisolone could be produced by the cows in stressful situations. In the present study, three dairy cows underwent intramuscular treatments with tetracosactide hexaacetate, a synthetic analogue of adrenocorticotropic hormone, to simulate stress. The animals were slaughtered at the end of the study. The results indicated that prednisolone could be detected occasionally in the non-stressful state, but was consistently found in the urine of stressed cows (concentrations ranged from 1.01 to 4.08 ng/mL). To confirm the stress condition, urinary cortisol and cortisone were also detected at high concentrations in the urine, typically at concentrations of hundreds of nanograms per millilitre. The results of this preliminary study did not reveal the metabolic pathway responsible for prednisolone but suggested that this corticosteroid could be produced endogenously.