In this work we present a theoretical analysis of the ratio method, a widely used technique for measuring intracellular calcium concentration, [Ca 2 + ] i , in isolated cells. From the ratio of fluorescence measured at two different excitation or emission wavelengths, [Ca 2 + ] i may be estimated from the equation: [Ca 2 + ] i = K d . β . (R - R m i n )/(R m a x - R). From this equation we determined the method sensitivity showing that its maximum is located at [Ca 2 + ] = K d . β . (R m i n /R m a x ) 1 2 i.e. for [Ca 2 + ] < K d . β We also analyzed the error propagation due to inaccuracies in the calibration parameters. The fluorescence phenomenon was described, aiming at providing a basis for the microscopic interpretation of the method and giving physical meaning to the calibration parameters. In this sense β, is shown to depend not only on the set-up, but also on the spectrum of the indicator for the particular sample studied. A new approach to estimate β with higher accuracy is also proposed. Experimentally obtained β values using this approach were not statistically different from those determined as F m i n 2 /F m a x 2 . A graphical interpretation of the method is presented to provide users of fluorescence systems with a simple technique to help understand equipment performance and design.