Isolated collagen fibrils from the bovine cornea and sclera were investigated by atomic force microscopy (AFM) in a non-contact mode. AFM imaging visualized the surface topography of both corneal and scleral collagen fibrils with quantitative information on their height and width. The corneal collagen fibrils had a height of 15.6±1.5nm and a D-periodicity of 63.9±0.5nm. On the other hand, the height and D-periodicity of scleral collagen fibrils were 74.2±55.7nm and 65.4±0.7nm, respectively. A periodic banding pattern of grooves and ridges was found in individual fibrils, although the groove depth was 2.81±0.29nm in the cornea and 5.47±0.66nm in the sclera. When collagen fibrils were treated with acetic acid, they swelled and untwisted into subfibrils. The AFM is useful to analyze surface morphology of collagen fibrils and their subfibrils at high resolution with quantitative information.