The interface of 10 or 25% (w/v) bovine serum albumin (BSA), pH 7, buffered solution against mineral or corn oil was studied with a Raman microscope. A gradient of distribution of protein and oil at the interface was observed. The difference spectrum obtained by subtracting the spectrum of mineral or corn oil from that of the BSA/oil interface indicated interactions involving different functional groups of the BSA and the oil molecules. Against mineral oil, the BSA spectrum showed reduced intensity of the tryptophan band at 750 cm(-1) and reduced intensity ratio of the tyrosine doublet at 850-830 cm(-1), indicating changes in the microenvironment of these hydrophobic residues. A negative band at 2850 cm(-1) indicated the involvement of the CH groups in the mineral oil. However, the amide regions, normally assigned to protein secondary structure, were not significantly changed. When the spectrum of BSA was subtracted from the BSA/mineral oil interface spectrum, the resultant difference spectrum showed changes of symmetric and antisymmetric CCC stretches at 980 and 1071 cm(-1), respectively. In contrast, the difference spectrum of BSA/corn oil interface - BSA showed a decrease of CH(2) symmetric stretching at 2850 cm(-1) and a decrease of unsaturated fatty acid hydrocarbon chain stretch at 3010 cm(-1). Raman spectroscopy is a useful tool to study the nature of protein-lipid interactions.