Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca2+ concentrations but inhibits it at micromolar Ca 2+ concentrations, indicating that binding of Ca2+ to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca2+ sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting ACRCaM probe displayed high-affinity binding to, and Ca2+-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca 2+, ACRCaM exhibited a substantial (> 50%) decrease in fluorescence (KCa = 2.7 ± 0.8 μM). A peptide derived from the RyR1 CaM binding domain (RyR13614-43) caused an even more pronounced Ca2--dependent fluorescence decrease, and a ≥ 10-fold leftward shift in its KCa (0.2 ± 0.1 μM). In the presence of intact RyR1 channels in SR vesicles, ACRCaM fluorescence spectra were distinct from those in the presence of RyR13614-43, although a Ca2+-dependent decrease in fluorescence was still observed. The KCa for ACRCaM fluorescence in the presence of SR (0.8 ± 0.4 μM) was greater than in the presence of RyR13614-43 but was consistent with functional determinations showing the conversion of ACRCaM from channel activator (apoCaM) to inhibitor (Ca 2+CaM) at Ca2+ concentrations between 0.3 and 1 μM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca2+ sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca2+ sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca2+.