It has been indicated in previous literature reports that divalent cations cannot directly condense DNA in aqueous solution. However, several studies have shown that divalent metal ions can induce a further condensation of DNA originally adsorbed on the surfaces of fluid cationic membranes. In this article, we examine the effect of divalent metal ions (Ca(2+) and Ni(2+)) on DNA condensed on the surfaces of a rigid cationic membrane formed by the mixture of cholesteryl 3-N-(dimethyl-aminoethyl) carbamate (DC-Chol) and 1,2-di(cis-9-octadecenoyl)-sn-glycero-3-phosphocholine (DOPC). Our results demonstrate that the divalent cations exert an electrostatic screening effect between DNA, where the in-plane spacing between DNA (d(DNA)) confined between the lipid bilayers gradually decreases with increasing cation concentration when the concentration exceeds a threshold value. The collapse transition, where d(DNA) drops discontinuously at a critical cation concentration, observed previously for the fluid membrane is not identified here, indicating the critical role of membrane flexibility in the two-dimensional condensation of DNA. (C) 2010 Elsevier Ltd. All rights reserved.