There is a paucity of information about total translation rate of vasopressin and oxytocin. Because the site of synthesis of the neurohypophysial hormones is anatomically separate from the site of storage, we were able to measure total translation by blocking transport of newly synthesized hormone and measuring accumulation in the areas of synthesis in the hypothalamus. Colchicine administered into the third ventricle in doses as low as 3.5 micrograms/rat blocked transport for 18 h. The linear increase in vasopressin and oxytocin content over 18 h indicated a stable rate of synthesis, which was 1.2 and 1.9 pmol/h for vasopressin and 1.4-2.5 pmol/h for oxytocin. The molar correlation for synthesis of total neurophysin to total hormone was 1.16. Infusion of oxytocin and vasopressin into rats indicated that this level of synthesis of hormone was essential under steady-state conditions to maintain plasma levels in the low physiological range of approximately 3 pg/ml for oxytocin and 1 pg/ml for vasopressin. The data on total synthesis of the neurohypophysial hormones provide a reference for studies in which physiological replacement is required and also provide the technique and base-line data to determine how translation of vasopressin and oxytocin is regulated when neurohypophysial function is altered.