The ability of [125I]calreticulin to bind to membrane fractions isolated from different muscle and non-muscle tissues was examined by a protein overlay technique. Specific [125I]calreticulin binding proteins were detected in rat liver smooth and rough endoplasmic reticulum and Golgi, in canine pancreatic microsomes, and in rabbit skeletal muscle sarcoplasmic reticulum. These proteins were confined only to membranes that contain calreticulin; they were not found in rat liver mitochondria or cytosol. [125I]Calreticulin binds to a 50-kDa protein and a number of lower M(r) (20,000-38,000) endoplasmic reticulum membrane proteins and to 30-kDa protein in skeletal muscle sarcoplasmic reticulum. Full-length calreticulin and the carboxyl-terminal region (C-domain) of the protein both competed with [125I]calreticulin for binding to the membrane proteins. Binding of [125I]calreticulin to pancreatic microsomes was also partially inhibited by the N-domain and to a lesser extent by the P-domain of the protein. We conclude that calreticulin interacts with the endoplasmic reticulum membrane proteins mainly through its carboxyl-terminal domain and that the endoplasmic and sarcoplasmic reticulum membranes may contain different calreticulin binding proteins.