Calcium uptake by rabbit skeletal sarcoplasmic reticulum (SR) is inhibited with an effective inactivation temperature (TI) of 37°C in EGTA with no effect on ATPase activity. Since the Ca-ATPase denatures at a much higher temperature (49°C) in EGTA, this suggests that a small or localized conformational change of the Ca-ATPase at 37°C results in inability to accumulate calcium by the SR. Using a fluorescent analogue of dicyclohexylcarbodiimide, N-cyclohexyl-N′-[4-(dimethylamino)-α-naphthyl]-carbodiimide (NCD-4), the region of the calcium binding sites of the SR Ca-ATPase was labeled. Steady-state and frequency-resolved fluorescence measurements were subsequently performed on the NCD-4-labeled Ca-ATPase. Site-specific information pertaining to the hydrophobicity and segmental flexibility of the region of the calcium binding sites was derived from the steady-state fluorescence intensity, lifetime, and rotational rate of the covalently bound NCD-4 label as a function of temperature (0-50°C). A reversible transition at ∼15°C and an irreversible transition at ∼35°C were deduced from the measured fluorescence parameters. The low-temperature transition agrees with the previously observed break in the Arrhenius plot of ATPase activity of the native Ca-ATPase at 15-20°C. The high-temperature transition conforms well with the conformational transition, resulting in uncoupling of Ca translocation from ATP hydrolysis as predicted from the irreversible inactivation of Ca uptake at 31-37°C in 1 mM EGTA. We conclude that an irreversible conformational change in the region of the calcium binding sites, of considerable lesser magnitude than unfolding, is responsible for the thermal inactivation of Ca uptake and uncoupling of Ca transport from ATP hydrolysis at elevated temperatures in EGTA.