AbstractThe time decay of fluorescence anisotropy for a dansylaziridine (DANZ) conjugate with Met‐25, which lies within the N‐terminal lobe of troponin C (TnC), shows at 10 and 25°C a longer correlation time characteristic of the entire molecule and a shorter correlation time arising from a more localized motion of the probe. In the absence of Ca2+, the amplitude of the shorter correlation time increases, suggesting an increased mobility of the probe. At 40°C, in both the absence and presence of Ca2+, a significant increase in probe mobility occurs. A 2,6‐toluidinyl naphthalene sulfonate (2,6‐TNS) complex with Ca2+‐liganded TnC shows only the longer correlation time at 12 and 25°C. An N‐(iodoacetylaminoethyl‐5‐naphthylamine‐1‐sulfonate) conjugate with Cys‐98 shows both a long and a short correlation time; the amplitude of the shorter correlation time is greater than for the DANZ conjugate. At 9, 25, and 40°C in the presence of Ca2+, and at 9°C in its absence, the magnitude of the long correlation time is consistent with motion of the entire molecule; at higher temperatures in the absence of Ca2+ it is substantially smaller, suggesting the presence of internal rotation. For Ca2+‐liganded TnC at temperatures of 25°C or lower, the results with all three labels are interpretable in terms of the crystallographic structure of TnC.