The results of work by several investigators indicate that crossbridge attachment serves as a positive feedback mechanism that transiently increases the Ca2+ affinity of troponin C (TnC) during each normal heartbeat. To monitor structural changes in the cardiac isoform of TnC (cTnC) associated with Ca2+ binding and crossbridge attachment in muscle, we labeled cTnC with the sulfhydryl-specific fluorescent probe 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid (IAANS). When IAANS-labeled cTnC (cTnCIAANS) was substituted for endogenous TnC, the fluorescence intensity of cardiac and skeletal muscle preparations increased substantially during rigor crossbridge attachment in the absence of Ca2+ (pCa 9.2). In cardiac muscle, the fluorescence signal increased the same amount in rigor and maximal activation, whereas in skeletal muscle, it was higher in rigor (rigor: cardiac and skeletal = 1; pCa 4.0: cardiac = 0.98 +/- 0.13, skeletal = 0.59 +/- 0.05). This indicates that crossbridge attachment alone is capable of influencing the structure of cTnCIAANS. Because the relative fluorescence intensity of cTnCIAANS was more sensitive to Ca2+ than was force in both preparations (cardiac: pCa50 fluorescence = 6.05 +/- 0.05, pCa50 force = 5.51 +/- 0.11; skeletal: pCa50 fluorescence = 5.94 +/- 0.13, pCa50 force = 5.65 +/- 0.14), we measured the Ca2+ sensitivity of the strong crossbridge attachment (sinusoidal stiffness was measured by imposing 1 kHz at 0.1-0.2% muscle length) in rat trabeculae.(ABSTRACT TRUNCATED AT 250 WORDS)