A high affinity [3H]ryanodine receptor has been solubilized from rabbit brain membranes and biochemically characterized. [3H]Ryanodine binding to rabbit brain membranes is specific and saturable, with a Kd of 1.3 nM. [3H]Ryanodine binding is enriched in membranes from the hippocampus but is significantly lower in membranes from the brain stem and spinal cord. Approximately 60% of [3H]ryanodine-labeled receptor is solubilized from brain membranes using 2.5% CHAPS and 10 mg/ml phosphatidylcholine containing 1 M NaCl. The solubilized brain [3H]ryanodine receptor sediments through sucrose gradients like the skeletal receptor as a large (approximately 30 S) complex. Solubilized receptor is specifically immunoprecipitated by sheep polyclonal antibodies against purified skeletal muscle ryanodine receptor coupled to protein A-Sepharose. [3H]Ryanodine-labeled receptor binds to heparin-agarose, and a protein of approximately 400,000 Da, which is cross-reactive with two polyclonal antibodies raised against the skeletal muscle ryanodine receptor, elutes from the column and is enriched in peak [3H]ryanodine binding fractions. These results suggest that the approximately 400,000-Da protein is the brain form of the high affinity ryanodine receptor and that it shares several properties with the skeletal ryanodine receptor including a large oligomeric structure composed of approximately 400,000-Da subunits.