Calsequestrin (CSQ)-binding 30-kDa protein in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle was purified from the junctional face membrane (JFM) in the SR. Analysis of proteins which bound to the affinity column conjugated with the purified 30-kDa protein was performed. As a result, in the heavy fraction of the SR (HSR), four proteins including CSQ proved to be adsorbed on the column, and in the JFM, one protein, whose molecular weight was about 25-kDa. was mainly adsorbed. Furthermore, the same 25-kDa protein was found to be adsorbed on the CSQ affinity column. This 25-kDa protein is probably the CSQ-binding 26-kDa protein (junctin) recently reported [Jones, L. R., et al. (1995) J. Biol. Chem. 270, 30787] judging from the molecular weight and the CSQ-binding property. These results suggest that three proteins, CSQ, 30-kDa protein, and 25-kDa protein, form a protein complex in the terminal cisternae of the SR.