Cisternal cerebrospinal fluid (CSF) of urethane anesthetized cats sampled at 30 min after the injection of 0.85M NaCl into the third ventricle of the brain, contains a frog bladder activity equivalent to 5000 ± 1000 (SE) μU/ml USP Posterior Pituitary Reference Standard. Trypsin inactivates 70% of the frog bladder activity, whereas tyrosinase destroys 80% of this activity. No frog bladder activity could be detected in CSF samples after the injection of 0.15M NaCl or 1.7M d—glucose into the third ventricle or when 0.85M NaCl was injected into the lateral ventricle of the brain. The frog bladder activity of cat CSF as well as its enzymatic characterization suggests that hypertonic saline injected into the third ventricle of the brain induced the release into the CSF of a basic peptide presumably identical with arginine vasotocin. (Endocrinology91: 825, 1972)