Electrostatic considerations predict that the binding of divalent cations to singly charged acidic lipids is likely to proceed with a 1:2 ion-to-lipid stoichiometry, but the fact that divalent cations at similar to 0.1 M concentrations reverse the sign of the negative surface potential of membranes provides evidence for 1:1 stoichiometry, In this work, analytic expressions are obtained for 1:1 and 1:2 binding constants and for the fractions of corresponding complexes based on extensions of the Gouy-Chapman-Stern theory and of Cohen-Cohen theory (J. A. Cohen and M. Cohen, Biophys. J. 36, 623, 1981), This approach relies on the ion concentration and the slope of the surface potential vs concentration curve at the charge reversal point. Application of this theory to previously published electrophoresis data on phosphatidylserine vesicles (S. McLaughlin, N. Mulrine, T. Gresalfi, G. Vaio, and A. McLaughlin, J. Gen. Physiol. 77, 445, 1981), varying the thickness of the shearing layer (delta) from 0 to 2 Angstrom, showed that (a) the 1:1 binding constants do not depend on delta and are similar to those determined in the original paper; (b) the 1:2 binding constants are extremely sensitive to delta : for Mg2+, Ca2+, Sr2+, and Ba2+, respectively, K-22 approximate to 25-35, 85-120, 110-150, and 190-270 M(-1) at delta = 0 and K-22 approximate to 2-3, 3-5, 5-7, and 10-15 M(-1) at delta = 2 Angstrom; (c) the fraction of lipid molecules involved in 1:2 complexes (theta) also strongly depends on delta : the values of theta calculated either at the charge reversal point or at the saturation of adsorption were 0.60-0.88 at delta = 0 and 0.22-0.55 at delta = 2 Angstrom. These results imply that the thickness of the shearing layer is a critical parameter for which the exact value is required to gain information on the ion binding mechanism from electrophoresis data. The present theory may also be applied to experimental results on surface potential vs concentration curves obtained by other methods.