Cotton (Gossypium hirsutum var. MD51) fiber cell walls were analyzed with an atomic force microscope (AFM) to determine the effect of chemical treatments on cell wall organization and topography. The results indicate that the AFM is an excellent tool to use to evaluate the morphology of the fiber surface and to quantitatively analyze the effect of each treatment. Treatment of fibers with 1% H₂O₂ had little effect on surface morphology. Alkali removed much of the cuticle, some primary wall components, and revealed mostly thin diameter microfibrils. Acidic Updegraff reagent fragmented the fibers, removed much of the cuticle, and revealed mostly thick microfibrils. The surface roughness of fibers treated sequentially with alkali and acid was quantitatively distinguishable from all other fiber types based on the standard deviation of the "Z" data (RMS), amplification of surface area (SAD), and integration of the scan line data (TP). Analysis of the fractal dimension enabled untreated and peroxide treated fibers to be clearly distinguished from the other fiber types. Segmentation of the fractal data revealed specific portions of the fractal dimension which were especially useful for defining the size of structures that differentiated fiber types. Areas containing microfibrils could be quantitatively differentiated from non-microfibrillar areas.