Arginine-8-vasopressin (AVP) levels were measured by a sensitive and specific radioimmunoassay (RIA) in plasma and cerebrospinal fluid (CSF) of three species man, dog, and rat (Wistar and the Brattleboro strain). Basal plasma values were 1.7 pg/ml in Wistar rat, and 2.4 pg/ml in dog. Pentobarbitone, used as anesthetic during collection of CSF from dog and rat, caused a significant rise of plasma AVP values in Wistar rats, but not in dogs. After withdrawal of CSF, the plasma AVP levels of Wistar rats were increased to 29.5 +/- 9.5 pg/ml, whereas the CSF levels from the same animals were 11.5 +/- 3.9 pg/ml. The response to the various stimuli was similar in Brattleboro rats, heterozygous for hereditary hypothalamic diabetes insipidus, and in Wistar rats. In Brattleboro rats, homozygous for hereditary hypothalamic diabetes insipidus, AVP was neither detectable in plasma nor in CSF. In dog and man, AVP levels in CSF samples were higher than in simultaneously obtained plasma samples. The possibility that AVP present in CSF, might be released directly from the synthetizing hypothalamic nuclei into the ventricular system is discussed.