The cell membrane complex in the cuticle of a human hair fibre or a rat whisker is composed of three layers, that is, β, δ and β layers. The X-ray diffraction technique is a powerful tool to investigate the pathway of aqueous molecules and ions across the cuticle. Small-angle scattering experiments using a micro X-ray beam, which can be applied to a cuticle of 5 µm thickness, provide the structural information on the cell membrane complex without interference from other structures. Taking into account the variation of thickness in the δ and β layers, the overall features of the diffraction profile in a small-angle region can be explained satisfactorily. The method makes it possible to analyse the structure of β, δ and β layers without assuming an ambiguous background in the diffraction profile, and was used for the analysis of a human hair fibre and a rat whisker. In a rat whisker, the X-ray diffraction was stronger and the variation in the layer thickness smaller than in a human hair fibre. This may be due to the fact that the rat whisker had not been washed with soap or cosmetically treated, whereas the variation may depend on the lipids or the proteins that each species naturally has. It is proposed that the method represents convenient tool for quantitative analysis to estimate the thickness of δ and β layers in the cell membrane complex.