Resonance-enhanced and normal vibrational Raman spectra were observed for both multilamellar and single-wall vesicle assemblies of dimyristoyl phosphatidylcholine containing amphotericin B, a channel-forming polyene antibiotic, and cholesterol. The decrease in the frequency of the polyene antibiotic C = C stretching mode at 1556 cm-1 and the increase in intensity of the C-C-H in-plane deformation mode at 1002 cm-1 indicate that amphotericin B is ordered in a lipid-cholesterol medium similarly to the solid, but is surrounded by a slightly more polar environment. The intensity of the C = C stretching mode I1556 decreases 4-fold during the broadened gel to liquid crystalline phase transition (16--32 degrees C) of dimyristoyl lecithin-cholesterol (4 : 1) multilayers. Other resonance-enhanced vibrations of amphotericin B exhibit similar behavior. For amphotericin B in pure dimyristoyl lecithin multilayer or vesicle systems, however, the vibrational intensity associated with the C = C stretching mode remains constant during the melting of lipid hydrocarbon chains. In addition, a third effect occurs in liquid crystalline egg lecithin-cholesterol (4 : 1, mol ratio) multilayers in which I 1556 first increases by 25% between 3 and 25 degrees C, in parallel with the loss of active channels, and then remains constant as the temperature increases from 25 to 42 degrees C. This latter intensity pattern is masked in the dimyristoyl lecithin-cholesterol system by the overwhelming effect upon the C = C mode from changes in the lipid chain packing characteristics which occur during the phase transition. The broadened phase transition in 4 : 1 dimyristoyl lecithin-cholesterol multilayers (16--32 degrees C), as followed by the ratio of intensities at 2880 and 2850 cm-1 (asymmetric and symmetric methylene C-H stretching modes, respectively) is slightly narrowed by the addition of amphotericin B, and effect from which a binding stoichiometry at 24 degrees of 1 : 1 amphotericin B: cholesterol is estimated. This stoichiometry was confirmed by differential calorimetric scans, which also show the presence of a peak proportional to cholesterol content. Raman I2880/2850 peak height ratios in pure dimyristoyl lecithin bilayers were increased over the 14--38 degrees C range by amphotericin B, a spectral effect which suggests an ordering of the lipid matrix perhaps as a consequence of the polyene binding to the bilayer surface. For bilayers containing cholesterol, the ratios of intensities of the 2935 cm-1 feature, composed mainly of acyl chain terminal methyl and underlying methylene C-H stretching modes, to the 2850 cm-1 feature are significantly increased by amphotericin B. This effect indicates that the antibiotic penetrates the bilayer in the lipid-sterol system.