The perturbations induced by melittin and cholesterol on the molecular organization of dimyristoyl-L-alpha-phosphatidylcholine (DMPC) bilayers, depending on the temperature and concentrations of both melittin and cholesterol, were studied using FT-IR spectrometry. Analysis of the amide I and amide II bands of melittin indicates that melittin exists as a monomer at all temperatures in the DMPC-melittin system (lipid:protein molar ratio R = 14:1) and in DMPC-cholesterol-melittin dispersions (R = 14 and 25) for low cholesterol concentrations (10 mol%). However in the DMPC-cholesterol-melittin systems with high cholesterol (20 mol%) and high melittin (R = 14) concentrations, melittin is found to be in a tetrameric form at T > T(m). The melittin-induced effects in DMPC bilayers containing a low melittin concentration (R = 25) are found to decrease on addition of cholesterol (10 or 20 mol%). Cholesterol, however, has the opposite effect in DMPC-cholesterol-melittin systems containing high cholesterol (20 mol%) and high melittin concentrations (R = 14), i.e., and increased lipid-protein interaction. Cholesterol- and melittin-induced effects on the spectral features of the phospholipid are discussed.