The characteristics of [(3)H]ouabain binding were studied in frontal cerebral cortex and striatum of ox brain. Specific [(3)H]ouabain binding reached equilibrium in approx. 40 min and was stable for at least 90 min. In frontal cortex membranes, Scatchard analysis revealed a single class of high-affinity binding sites with an apparent dissociation constant (K(d)) of 14 +/- 2.0 nM and a maximal number of binding sites (B(max)) of about 44.7 +/- 3.8 pmol/mg protein. A similar monophasic Scatchard plot was found in striatal membranes. The density of binding sites was of the same magnitude in these two cerebral areas, but striatal sites showed a lower binding affinity. The addition of K(+) or of Ca(2+) to the assay also reduced specific binding and the rank order of inhibitory potencies was similar to that previously reported for inhibition of [(3)H]ouabain binding to rat brain. All cardiac glycosides which were so far tested inhibited specific [(3)H]ouabain binding to frontal cortex membranes. Ouabain was the most potent compound tested (IC(50) 18.3 nM) while digoxigenin was the least potent (IC(50) 112.3 nM). Theophylline was a very weak inhibitor of specific binding. These results suggest the presence of high-affinity [(3)H]ouabain binding sites in ox brain membranes, and are related to previous reports for ouabain binding to human and rat brain.