VP3(110-121), a peptide from the exposed VP3 capsid protein of hepatitis A virus, has been shown earlier to bind at pH 7.4 to bilayered vesicles containing the major phospholipids found in the membrane of hepatocytes and to cause aggregation and fusion of these vesicles by subsequent acidification. To deepen the understanding of the interaction of VP3(110-121) with the lipid membrane, in this paper we report a detailed study of the miscibility properties and behavior of mixed monomolecular films of these major lipid components at the air/water interface. The miscibility of these components in mixed monolayers was determined with the Langmuir film technique and the excess free energy of mixing (Delta G(M)(ex)) and interaction energies were calculated. Results show that all the components are miscible in all ratios and indicate the presence of interactions of low energy between them at certain mole fractions. Next, we have measured the insertion of VP3(110-121) into spread monolayers of these components at mole ratios that closely resemble the hepatocyte membrane. Insertion was measured as a function of the lateral packing of the monolayer. Three compositions with different net charge were selected: 1-palmitoyl-2-oleoylphosphatidylcholine:cholesterol:sphingomyelin: 1,2-dipalmitoylphosphatidylethanolamine (POPC/CH/SM/DPPE) (35: 24:31:10), a mole ratio similar to the hepatocyte membrane, and its mixtures with a cationic lipid (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) POPC/CH/SM/DPPE/DOTAP (32:21:28:9:10) and with an anionic lipid (L-alpha-phosphatidyl-L-serine from bovine brain, PS) POPC/CH/SM/DPPE/PS (32:21:28:9: 10). Results indicate that the interaction of VP3(110-121) has both hydrophobic and electrostatic components, with insertion in all three monolayers but with a stronger interaction with the cationic interface, with higher increases in surface pressure and faster insertion rates. The lowest affinity of binding corresponds to anionic PS-containing films, whereas interaction with zwitterionic POPC/CH/ SM/DPPE films is intermediate. This is expected due to the anionic net charge of this amphiphilic peptide at the pH of the experiments.