The present study was designed to analyze the subfibrillar structure of corneal and scleral collagen fibrils by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Isolated collagen fibrils of the bovine cornea and sclera were fixed with 1% OsO4, stained with phosphotungstic acid and uranyl acetate, dehydrated in ethanol, critical point-dried, metal-coated, and observed in an in-lens type field emission SEM. Some isolated collagen fibrils were fixed with 1% OsO4, dehydrated, critical point-dried and observed without metal-coating in an AFM. Isolated collagen fibrils treated with acetic acid were also examined by SEM and AFM. SEM and AFM images revealed that corneal and scleral collagen fibrils had periodic transverse grooves and ridges on their surface; the periodicity (i.e., D-periodicity) was about 63 nm in the cornea and about 67 nm in the sclera. Both corneal and scleral collagen fibrils contained subfibrils running helicoidally in a rightward direction to the longitudinal axis of the fibril; the inclination angle was about 15 degrees in the corneal fibrils and 5 degrees in the scleral fibrils. These findings indicate that the different D-periodicity between corneal and scleral fibrils depends on the different inclinations of the subfibrils in each fibril. The present study thus showed that corneal collagen fibrils differ from scleral collagen fibrils not only in diameter but also in substructure.