Fourier transform infrared microscopy has been applied to the characterization of hypercholesterolemic rabbit liver. A combination of the traditional "group frequency" approach to spectral interpretation and the technique of functional group mapping was applied to aid understanding of the spectral changes observed between normal and hypercholesterolemic liver. Using this approach, spectral differences could be attributed to the accumulation of cholesterol esters (most likely in the form of lipoprotein complexes) in the hypercholesterolemic liver. A novel approach, spectral clustering, was used to identify regions of hypercholesterolemic liver tissue which possessed similar spectral (and by inference similar biochemical/histological) properties. Using this approach, a complex data set consisting of 400 discrete spectra acquired from an area of tissue approximately 1 x 1 mm could be reduced to five types of spectra, and the distribution of each type of spectrum within the tissue analyzed.