High affinity [3H]ryanodine binding sites were characterized in P1 (crude nuclear), P2 (mitochondrial/synaptosomal) and P3 (microsomal) subcellular fractions of rat brain. Binding in each of the fractions was highest at 37 degrees C and pH 8-9, optimal in the presence of 100 microM Ca2+, 550 microM ATP and 1.0 M KCl, and increased linearly as a function of protein. Saturation analyses revealed a single class of binding sites with mean KD values (nM) of 8.9, 1.6 and 5.7 and Bmax values (fmol/mg protein) of 122, 69 and 106 for the P1, P2 and P3 fractions, respectively. The levels of [3H]ryanodine binding in P1 and P2 fractions of 4 brain regions were fairly uniform while those in P3 fractions were 5-fold greater in cerebral cortex than in the other areas examined. By autoradiography, a high concentration of [3H]ryanodine binding sites was seen in the dentate gyrus and CA3 subregions of the hippocampus. The results suggest that [3H]ryanodine binding sites, perhaps similar to [3H]ryanodine receptors in muscle, are associated with various subcellular structures and are heterogeneously distributed in the CNS.