A procedure is described for collection of blood from the confluens sinuum of the rat. Melatonin concentration, measured by RIA, in plasma of the confluens was found to be significantly higher than that in peripheral plasma. Melatonin levels in cerebrospinal fluid of the cisterna magna were always lower than those in plasma and exhibited no significant changes which paralleled those occurring in plasma with darkness. It is concluded that, in the rat, the major route of secretion of melatonin by the pineal gland is into the blood of the confluens sinuum.