Ca²⁺-induced Ca²⁺ release (CICR) in the heart involves local Ca²⁺ signaling between sarcolemmal L-type Ca²⁺ channels (dihydropyridine receptors - DHPRs) and type 2 ryanodine receptors (RyR2s) in the sarcoplasmic reticulum (SR). We reconstituted cardiac-like CICR by expressing a cardiac dihydropyridine-insensitive (T1066Y/Q1070M) ₁-subunit (1C_YM) and RyR2 in myotubes derived from RyR1 knockout (dyspedic) mice. Myotubes expressing 1C_YM and RyR2 were vessiculated and exhibited spontaneous Ca²⁺ oscillations that resulted in chaotic and uncontrolled contractions. Co-expression of FKBP12.6 (but not FKBP12.0) with 1C_YM and RyR2 eliminated vessiculations and reduced the percentage of myotubes exhibiting uncontrolled global Ca²⁺ oscillations (63% and 13% of cells exhibited oscillations in the absence and presence of FKBP12.6, respectively). 1C_YM/RyR2/FKBP12.6-expressing myotubes exhibited robust and rapid electrically-evoked Ca²⁺ transients that required extracellular Ca²⁺. Depolarization-induced Ca²⁺ release in 1C_YM/RyR2/FKBP12.6-expressing myotubes exhibited a bell-shaped voltage dependence that was 4-fold larger than that of myotubes expressing 1C_YM alone ((F/F)_max was 2.10±0.39 and 0.54±0.07 respectively), in spite of similar Ca²⁺ current densities. In addition, the gain of CICR in 1C_YM/RyR2/FKBP12.6-expressing myotubes exhibited a non-linear voltage dependence, being considerably larger at threshold potentials. We used this molecular model of local _1C-RyR2 signaling to assess the ability of FKBP12.6 to inhibit spontaneous Ca²⁺ release via a phosphomimetic mutation in RyR2 (S2808D). Electrically-evoked Ca²⁺ release and the incidence of spontaneous Ca²⁺ oscillations did not differ in wild-type RyR2- and S2808D-expressing myotubes over a wide range of FKBP12.6 expression. Thus, a negative charge at S2808 does not alter in situ regulation of RyR2 by FKBP12.6.