We have measured the binding of two radioiodinated monoclonal anti-dinitrophenyl antibodies (IgE and IgG2a) to two dinitrophenylated lipid haptens in lipid bilayer membranes having various compositions and physical properties. These antibodies bind strongly to the lipophilic dinitrophenyl group in some membranes. Dimyristoylphosphatidylcholine and dipentadecanoylphosphatidylcholine containing 2 mol % dinitrophenyl lipid hapten bind anti-dinitrophenyl antibodies below the chain-melting transition temperatures of these lipids (22 and 35 degrees C, respectively) but not above these temperatures. Evidently, the lipophilic dinitrophenyl group is partially or completely buried in the hydrophobic region of these bilayers at temperatures above the chain-melting transition temperatures. The inclusion of increasing concentrations of cholesterol in such membranes (e.g. in dimyristoylphosphatidylcholine at 37 degrees C) results in a marked enhancement of antibody binding. It was found that a third lipid hapten containing the dinitrophenyl group does not show this strong dependence of antibody binding on the physical state of the lipid membrane. The weak immunologic degranulation of rat basophil leukemia cells by dimyristoylphosphatidylcholine membrane targets at 37 degrees C can be attributed to a weak binding of anti-dinitrophenyl IgE to these membranes (Balakrishnan, K., Hsu, F. J., Cooper, A. D., and McConnell, H. M. (1982) J. Biol. Chem. 257, 6427-6433). However, if the antibody is first allowed to bind to this membrane below the lipid chain-melting transition temperature, these IgE-coated membrane targets are very effective in releasing serotonin from the rat basophil leukemia cells when the temperature is raised to 37 degrees C.