Accurate estimates of cytosolic free Ca2+ with fluorescence indicators are dependent on the determination of the in situ dissociation constants (kd) of the intracellular dyes and the correction for background fluorescence. The in situ dissociation constant for Ca2+ and indicator dye Fura-2/AM varies significantly from the in vitro published values due to differences in ionic strength, pH, viscosity and Ca2+ buffering by intracellular lipids and proteins. During the course of a measurement, background fluorescence changes may occur as the result of endogenous fluorescent compounds and compartmentalized Fura-2 indicator. The in situ dissociation constant value was determined for human astrocyte cell line U373-MG by creating several known intracellular Ca2+ concentrations while measuring total fluorescence and background fluorescence values for each. The background fluorescence was not constant, rather it demonstrated a linear relationship with the free cytosolic Ca2+ concentrations and total fluorescence intensities. The Ca2+ dependent and total fluorescence dependent background was expressed as a linear equation and subtracted appropriately from the total intensity measurements. The in situ dissociation constant was determined to be 3-fold greater than in vitro measurements after the background was corrected. The experimentally determined standard linear equations for background quantitation and the in situ dissociation constant for this line produce accurate cytosolic free Ca2+ estimates.