Excitation-contraction coupling in muscle cells requires an interaction between L type calcium channels of surface membrane/T tubules (the dihydropyridine receptors, DHPRs) and the calcium release channels or ryanodine receptors (RyRs). of the sarcoplasmic reticulum(SR), Immunolabelling with specific antibodies shows that the two types of calcium channels form part of discrete macromolecular complexes, called calcium release units, that are located at the surface membrane and/or along the T tubules. The complexes include several SR proteins (calsequestrin, triadin, junctin), RyR associat ed proteins (Homer, FKBP12); the DHPRs and a docking protein that connects SR and surface membranes (junctophilin). Skeletal and cardiac muscles contain three types of RyRs: (RyR1 and RyR3 for skeletal; RyR2 for cardiac) and two forms of the a1 subunit of DHPRs (a1 s and a1c). The location of these isoforms and their positioning relative to each other are quite informative in regard to their functional interactions and to the role they play in excitation-contraction coupling. We have explored this question in a variety of native muscles and in cells engineered for null mutations of either channel and induced to express chimeric forms of the cardiac and isoforms. a1sDHPR and RyR1 are essential components of the skeletal muscle e-c coupling machinery and they are located in coextensive and interlocked arrays. Within the arrays, groups of four DHPRs (forming a tetrad) are located at predetermined specific locations relative the four subunits of every other RyR1 in the array. Grouping of DHPRs into tetrads requires the presence of RyR1. This association provides the structural framework for the reciprocal signalling that is known to occur between the two types of channels. RyR3 are present in some but not all skeletal muscles, always in association with RyR1, and in ratios as high as 1:1 ratio with the latter. RyR3 neither induce formation of tetrads by DHPRs, nor sustain e-c coupling. In CRUs of muscle fibres, RyR3 are located in a parajunctional position, in proximity of the RyR1-DHPR complexes. It is thus expected that they may be indirectly activated by calcium liberated via the RyR1 channels. RyR2 channels, the only isoform present in cardiac muscle, have two locations. One is at CRUs formed by the association of SR cisternae with domains of surface membrane and/or T tubules that contain DHPRs. In these cardiac CRUs, RyR2 and a1cDHPR are in proximity of each other, but not closely linked. This is in keeping with the proposed indirect DHPR-RyR interaction during e-c coupling of cardiac muscle. A second location is on SR cisternae that are not directly linked to surface membrane/T tubules. The RyR2 in these cisternae, which are often several microns away from any DHPRs, must necessarily be activated indirectly, Cardiac/skeletal RyR chimerae expressed in RyR null cells and a1s/a1c DHPR chimerae expressed in a1 null skeletal muscle cells show that DHPR tetrads are always induced by the presence of Either DHPR or RyR chimerae that restore skeletal-type e-c coupling. Several regions of RyR and at least one critical region of DHPRs are necessary for tetrad formation, but the molecular domains necessary for tetrad formation are not exactly the same as those required for an inter-molecular interaction. Animal experimentation conformed to local standards.