Although the RNA for the third isoform (Ryr3) of ryanodine receptor (RyR), a Ca2+ release channel, is detected in specific regions of mammalian brain, little is known about the protein. We investigated Ryr3 in rabbit brain, using an antibody raised against the synthetic peptide corresponding to amino acid sequence 4375-4387 of rabbit Ryr3, the homologue of bullfrog beta-RyR. The antibody which reacted with bullfrog beta-RyR, but not with the other isoforms, Ryr1 or Ryr2, specifically precipitated a single polypeptide from rabbit brain microsomes having a size similar to beta-RyR. Sucrose gradient ultracentrifugation revealed that Ryr3 forms a homotetramer, as true of the other isoforms. Being consistent with the distribution of its RNA, Ryr3 was abundantly expressed in hippocampus, corpus striatum, and diencephalon. Ryr3 demonstrated Ca2+-dependent [3H]ryanodine binding, and caffeine increased its Ca2+ sensitivity. The Ca2+ sensitivity of Ryr3 was also enhanced in a medium containing 1 m NaCl, as observed with beta-RyR. [3H]Ryanodine binding gave an estimate of Ryr3 which would be only 2% or less of total RyR in rabbit brain. These results confirm the expression of functional Ryr3 in mammalian brain which is similar to nonmammalian beta-RyR.