The formation of acellular cementum and the deposition of [3H]mannose-labeled extracellular matrix were studied in 14-day-old Sprague-Dawley rats. The sequential events of cementogenesis and periodontal ligament formation observed by light and electron microscopy were described from the stage of an intact root sheath to postcementogenesis. Ultrastructural examination of cementoblasts and periodontal ligament fibroblasts revealed [3H]mannose labeling of the Golgi apparatus at 10 minutes, collagen secretion granules at 30 minutes, and the extracellular matrix beginning at 30 minutes. The extracellular matrix between cementoblasts and dentin was heavily labeled at 1 and 4 hours. Newly formed principal fibers of the periodontal ligament were also heavily labeled at 4 hours. Fully differentiated cementoblasts exhibited the largest sectional profiles and the highest number of silver grains per unit area of cytoplasm. The morphologic and radioautographic data suggest that during the formation of acellular cementum, the cementoblast phenotype is expressed for a short period of time, after which cementoblasts appear to mix with the fibroblasts of the periodontal ligament.