X-ray reflectivity and fluorescence near total reflection experiments were performed to examine the affinities of divalent ions (Ca2+ and Ba2+) from aqueous solution to a charged phosphatidic acid (PA) surface. A phospholipid (1,2-dimyristoyl-sn-glycero-3-phosphate, DMPA), spread as a monolayer at the air/water interface, was used to form and control the charge density at the interface. We find that, for solutions of the pure salts (i.e., CaCl2 and BaCl2), the number of bound ions per DMPA at the interface is saturated at concentrations that exceed 10(-3) M. For 1:1 Ca2+/Ba2+ mixed solutions, we find that the bound Ca2+/Ba2+ ratio at the interface is 4:1. If the only property determining charge accumulation near PA were the ionic charges, the concentration of mixed Ca2+/Ba2+ at the interface would equal that of the bulk. Our results show a clear specific affinity of PA for Ca compared to Ba. We provide some discussion on this issue as well as some implications for biological systems. Although our results indicate an excess of counterion charge with respect to the surface charge, that is, charge inversion, the analysis of both reflectivity and fluorescence do not reveal an excess of co-ions (namely, Cl- or I-).