The effect of intraventricular and intravenous (i.v.) hypertonic saline on plasma and perfusate arginine vasopressin (AVP) and oxytocin (OT) levels was determined. A push-pull technique was used to sample third ventricular cerebrospinal fluid (CSF) in the conscious unrestrained rat. Intraventricular perfusion of hypertonic saline caused a 6.1- and 4.2-fold increase in perfusate levels of AVP and OT. Plasma levels with the same stimulus increased 3.5-fold for AVP and 3.4-fold for OT. Peak levels of the peptides occurred at 30 and 60 min in the CSF perfusate and plasma, respectively. Thus, the central peptide response occurred earlier and was of a greater magnitude than the systemic one. Intravenous hypertonic saline administration caused a rapid (15 min) increase in plasma AVP and OT, changes of 12.6- and 22.9-fold. Perfusate AVP did not change with i.v. hypertonic saline while OT was increased 10-fold. Two types of recovery experiments were performed (1) in which the rate of disappearance of 125I-labeled peptides from CSF was determined and (2) in which total body water was labeled with 3H2O and the amount of dilution determined. Both techniques showed that recovery of endogenous CSF in the push-pull perfusate was approximately 15%. Thus, the perfusate peptide levels represent an underestimation of in vivo secretion. Osmotic stimuli elicit specific changes in the third ventricular levels of AVP and OT. The differential response in the two hormones to i.v. hypertonic saline shows a uniqueness in the mechanisms controlling the central secretion of AVP and OT.