The present study describes the ultrastructure of the innermost layer of cementum (ILC) in the rat molars and discusses its developmental process and calcification mechanisms. The following points are the main results of this study: 1. The ILC is a thin layer, about 2.0?m thick, intensely stained with hematoxylin but not stained by silver impregnation. 2. Electron microscopically, it is composed of substances stained with ruthenium red and chromic phosphotungstic acid presumed to be proteoglycans and a few thin collagen fibrils. 3. Periodontal fibers penetrate the ILC only a short distance, and do not reach the root dentin surface. 4. The ILC begins to form on the root dentin surface, just after the disintegration of Hertwig's epithelial root sheath. At the same time, matrix vesicles and spherical bodies, which may be derived from the matrix vesicles, appear on the surface of the developing ILC. 5. Dental sac cells show higher cell activities than the epithelial sheath cells. Observations support the view that the dental sac cells secrete the ruthenium red positive material. 6. On the basis of the above findings, the ILC is suggested to be formed by the dental sac cells and calcified by the matrix vesicles derived from these cells. 7. The ILC can be regarded as a specialized Cementum between the root dentin and the cementum in the strict sense, serving the connection of the two tissues.