A 1 2-kDa immunophilin (FKBP12)is an integral component of the skeletal muscle ryanodine receptor (RyR). The RyR is a hetero-oligomeric complex with structural formula (FKBP)4(Ryrl)4, where Ryrl is the 565-kDa product of the Ryrl gene. To aid in the detection of the immunophilin's location in the receptor, we exchanged the FKBP12 present in RyR-enriched vesicles derived from sarcoplasmic reticulum with an engineered construct of FKBP12 fused to glutathione S-transferase and then isolated the complexes. Cryoelectron microscopy and image averaging of the complexes (in an orientation displaying the RyR's fourfold symmetry) revealed four symmetrically distributed, diffuse density regions that were located just outside the boundary defining the cytoplasmic assembly of the RyR. These regions are attributed to the glutathione transferase portion of the fusion protein because they are absent from receptors lacking the fusion protein. To more precisely define the location of FKBP1 2, we similarly analyzed complexes of RyR containing FKBP1 2 itself. Apparently some FKBP is lost during purification or storage of the RyR because, to detect the receptor-bound immunophilin, it was necessary to add FKBP1 2 to the purified receptor before electron microscopy. Averaged images of these complexes showed a region of density that had not been observed previously in images of isolated receptors, and its position, along the edges of the transmembrane assembly, agreed with the position of the FKBP12 deduced from the experiments with the fusion protein. The proposed locations for FKBP1 2 are about 10 nm from the transmembrane baseplate assembly that contains the ion channel of the RyR.