Troponin (Tn) is the molecular switch of striated muscle contraction. It is a heterotrimeric protein consisting of a Ca2+ binding subunit (TnC), an inhibitory subunit (TnI), and a thin filament anchoring subunit (TnT). Despite the vast amount of x-ray and NMR structural data available for Tn and its subunits, defining the molecular details of the conformational changes triggered by Ca2+ binding in the presence of its binding partners of the thin filament, is still needed and experimentally remains a challenge. Additionally, isoform differences between the Tn subunits may play a more significant role for switching muscle between the relaxed and active states than traditionally believed, with phosphorylation of the TnI subunit possibly playing a key regulatory role. Here we have used continuous wave (CW) and Double Electron-Electron Resonance (DEER) pulsed EPR spectroscopy to measure distances between spin labels placed on TnI and TnC to track the movement of the functionally important TnI regulatory ‘switch’ region in whole muscle filaments. Under conditions of high Ca2+, interspin distances were ‘short’ (9-10 Å) with narrow distance distribution widths (<8 Å) indicating the close interaction of the switch region with the N-lobe of TnC. Additional longer interspin distance populations with notably broader distance distribution widths (14-29 Å) were also detected by DEER. Upon Ca2+ removal, the interspin population shifted toward the longer distances, indicating the release of the switch region from TnC and an overall increase in disorder for this region. The results suggest that the close proximity of the TnI switch region to TnC in the cardiac isoform is necessary for promoting the interaction between the regulatory switch helix with the N-lobe of cardiac Troponin C, which, unlike the skeletal isoform, is largely in a closed conformation. The effect of cardiac specific phosphorylation on this mechanism will also be presented.