Fourier-transform infrared spectroscopy was used to examine the secondary structure of rabbit liver cytochrome b₅ and the polar and nonpolar domains of the protein. The data for both the ploar and nonpolar domains agree well with those previously obtained by other physical techniques. In particular it was found that the nonpolar membrane-binding domain was predominantly α helix and the polar domain was also highly helical, but not all α helix. The independence of the two domains in the whole molecule was, in general, confirmed by the additivity of the spectra of the two domains. The small differences that were seen inidicate the there is a loss of α helix when the protein is cut it into the two domains. In addition, there appears to be slight difference in the exposure to solvent of the amide NH groups in the α-helical portion of the nonpolar domain when it was examined in isolation.