Vasopressin (AVP)-binding proteins were obtained from rat brain and the anterior and posterior lobes of bovine pituitary glands by (a) preparation of crude membranes, (b) solubilization of membrane proteins, (c) passage through an affinity column containing immobilized AVP, and (d) elution from the column with excess AVP. Gel electrophoresis revealed protein bands of 55 and 62 kilodaltons in rat brain, bovine posterior lobe and, to a far lesser extent, in the anterior lobe, which were similar to those previously identified in rat brain to be associated with AVP binding. Immunoblotting demonstrated that the 55 kilodalton bands of rat brain and bovine pituitary gland were selectively immunoreactive with an AVP anti-idiotype antibody. In addition, immunoreactivity occurred with a 62 kilodalton component of rat brain.