Specific binding sites for arginine vasopressin (AVP) were demonstrated on rat brain membranes using [3H]AVP of high specific activity. At pH 7.4 in the presence of 5 mM MgCl2, one class of sites was measured with a KD of 0.56 nM and a Bmax of 4.3 fmol/mg protein. At pH 8.0 in the presence of MgCl2, two distinct sites were observed, having KD values of 0.42 and 13 nM and Bmax values of 5.6 and 68 fmol/mg protein, respectively, and similar results were obtained at pH 7.4 after repeatedly freezing and thawing the membranes. Binding increased with pH, apparently representing increased occupancy of the high capacity, lower affinity site. Binding to the lower affinity site was also enhanced by freezing and thawing membranes, or by adding 5 mM NiCl2 or 10 microM ZnCl2 to the incubation medium, whereas binding to the high affinity site was dependent on the addition of Mg. AVP was over 35 times more active in displacing 0.4 nM AVP than oxytocin or arginine-vasotocin, and 10,000 times more active than somatostatin. A number of other peptides had no effect on [3H]AVP binding at concentrations up to 10(-5) M. Autoradiography and regional dissection studies revealed a marked concentration of high affinity AVP-binding sites in the supraoptic and paraventricular nuclei of the hypothalamus, and Mg significantly enhanced the binding in these regions.