Two experiments were designed to determine if melatonin concentrations differ between jugular veins. In a first experiment, blood was collected continuously every 30 min from each jugular during 12-h from 6 ewes. In a second experiment, 100 ewes were sampled twice at night simultaneously from the two jugular veins. In both experiments, mean melatonin concentrations were similar between right and left jugulars. However, within individuals, large differences were observed between the two sides (p < 0.001). This difference was stable over time and the higher side varies among individuals. The concentrations of prolactin and oxytocin, measured in the same samples did not show such differences. This observation raises the question of the origin of this phenomenon. Moreover, it has important implications for studies in which melatonin concentrations need to be assessed accurately. Indeed, a reliable quantitative assessment of melatonin production by the pineal gland requires sampling from both jugular veins. (C) 2009 Elsevier Ltd. All rights reserved.