Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca2+release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca2+release events (Ca2+sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca2+release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca2+sparks, Ca2+-induced Ca2+release, and Ca2+waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6+/+, and RYR3+/+mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca2+sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca2+sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca2+-induced Ca2+release was similarly augmented in FKBP12.6+/+, but not in RYR3 null cells relative to wild-type. Finally, Ca2+wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca2+wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca2+sparks in smooth muscle, and the finding of normal Ca2+sparks and CICR in RYR3 null mice, indicate that Ca2+release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca2+sparks, and associated STOCs, in smooth muscle.