The 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 25Β°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 25Β°C. A n N-(iodoacetylaminoethyl-5-naphthylamine-l-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 40Β°C in the presence of Ca2', and at 9Β°C in its absence, the magnitude of the long correlation time i s 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 25Β°C or lower, the results with all three labels are interpretable in terms of the crystallographic structure of TnC.