Skeletal muscle contraction is regulated by calcium-dependent changes in the structure and thin-filament location of troponin and tropomyosin. The structural changes in the isolated calcium-binding subunit of troponin (TnC) are well characterized, but those of TnC in the native thin filament are much less clear. We measured the in situ orientation of the C-terminal lobe of TnC (CTnC) by polarized fluorescence from bifunctional rhodamine (BR) probes cross-linking pairs of cysteines at TnC residues 96-103, 116-123, 132-139, and 119-135. Each BR-labeled TnC was exchanged into single permeablized fibers from rabbit psoas muscle, and polarized fluorescence from the BR-TnCs was measured during relaxation and maximal calcium activation. The orientation distribution of CTnC with respect to the thin filament axis was calculated by maximum entropy analysis using the in vitro structure of CTnC in the troponin core complex (Vinogradova et al. (2005) PNAS102:5038-5043). The peak angle between E helix of CTnC and the filament axis was 49° in relaxed muscle and 64° during maximal activation. Comparison with the results of our previous study of the orientation of the N-terminal lobe of TnC (Ferguson et al. (2003) Mol. Cell11: 865-874) suggests that the central D/E helix of TnC is bent by about 30° in relaxed muscle and becomes straight during maximal activation.Supported by MRC (UK).