Wool fibre surfaces have been treated by solvent cleaning which leaves the native covalently bound surface lipid layer intact, and by alcoholic alkali which removes the lipid layer. The resultant surfaces have been analysed by atomic force microscopy (AFM), with particular emphasis on force-distance (F-d) methods. Methodologies were developed for investigation in situ in water of both the surface topography and the characteristics of the lipid layer. Longitudinal surface texturing was resolved in images of wool fibre surfaces in air; the texturing remained prominent after exposure to water. High resolution F-d curves revealed features associated with the lipid layer. A simple formalism was used to show that the layer had a thickness of a few nm, and an effective stiffness of some 0.12ᰮ01 N/m. Strong adhesive interactions, equivalent to a pressure of 0.1 MPa, acted on the tip at the tip-to-substrate interface. The methodology and formalism are likely to be relevant in the broad field of thin-film analysis and for fibre technology.