Antisense oligonucleotides inhibit the expression of complementary target sequences with extraordinary selectivity and thus have great potential for development of novel therapeutics in many critical areas, including cancer and infectious disease. However, the mechanism by which oligonucleotides pass through the cell membrane and reach the site of action is not well understood. In this study, phospholipid monolayers were used as models of cell membranes. Using a Langmuir film balance, the effects of ions, oligonucleotides, and other polyions (polylysine) on the isotherms of phospholipid monolayers were determined. Differences were observed in the coexistence region between liquid expanded and condensed phases. These results indicate that calcium or other divalent cations may mediate interaction of oligonucleotides with DPPG or other anionic membrane components.