Linkage between the high-capacity Ca 2 + -binding protein calsequestrin and the ryanodine receptor is proposed to be essential for proper Ca 2 + -release during skeletal muscle excitation-contraction coupling. However, no direct biochemical evidence exists showing a connection between these high-molecular-mass complexes in native skeletal muscle membranes. Here, using immunoblot analysis of chemically crosslinked membrane vesicles enriched in triad junctions, we have demonstrated that a very close neighborhood relationship exists between calsequestrin and the ryanodine receptor in both main fiber types. Hence, the luminal Ca 2 + -reservoir complex appears to be directly coupled to the membrane Ca 2 + -release complex and oligomerization seems to be of functional importance.