In skeletal muscle, excitation-contraction (EC) coupling and retrograde signaling are thought to result from direct interactions between the ryanodine receptor (RyR1) and the α1 subunit of the dihydropyridine receptor (α1S). Previous work has shown that the s53 region of α1S (residues 720-765 in the II-III loop) and regions R10 (1635-2636) and R9 (2659-3720) of RyR1 are involved in this signaling. Using the yeast two-hybrid system, we here report an interaction between s53 and the sR16 region of RyR1 (1837-2168, within R10), whereas no interaction was seen using upstream residues of the α1S II-III loop (s31, 666-709). The specificity of the s53-sR16 interaction was tested by using fragments of the cardiac RyR (RyR2) and DHPR (α1C) that correspond to sR16 and s53, respectively. No interaction was observed for sR16 × c53 (α1C 850-897), but weak interaction was occasionally observed for s53 × cR16 (RyR2 1817-2142). To test the functional significance of the s53 × sR16 interaction, we expressed in dyspedic myotubes a chimeric RyR (chimeraR16) in which sR16 was substituted for the corresponding region of RyR2. ChimeraR16 was found to mediate weak skeletal-type EC coupling. To test the necessity of sR16 sequence for coupling, we used "chimeraR16-rev," in which sR16 and a small upstream region of RyR1 were replaced by RyR2 sequence. ChimeraR16-rev did not differ from RyR1 in its ability to mediate EC coupling. Thus, interaction between residues 720-765 of α1S and residues 1837-2168 of RyR1 appears to contribute to but is not essential for EC coupling in skeletal muscle.