The interaction of Fura-2 with Ca2+ is studied using steady fluorescence technique.The effect of pH on the spectra behavior of Fura-2 in the presence of Ca2+ is investigated,the excitation maxima (340 nm) and the isobestic point (360 nm) for the fluorescence spectra of Fura-2 depend on pH.At different temperatures the apparent dissociation constants (Kd) of Fura-2-Ca2+ complex are examined,Kd is found to decrease with increasing temperatures (20 ℃,37 ℃,50 ℃) and △H is calculated to be 21.16 kJ/mol by using the Vant Hoff equation at pH7.4 for all the temperatures tested.The determination of intracellular Ca2+ concentration ([Ca2+]i) in lymphocyte is developed by using Fura-2 as a fluorescence probe in the presence of Cefotaxime at 37 ℃ and pH 7.4.