Phospholipids have been identified in enamel and dentin. Before demineralization, a group of phospholipids extracted by lipid solvents was associated with cell membranes and is therefore closely related to cell growth and intracellular regulations. After demineralization, a second group of phospholipids, associated with the extracellular matrix, was extracted; this group is probably linked to the mineralized phase. Using imidazole-osmium tetroxide fixation of rat incisors, we stained cellular unsaturated fatty acids, so that we could visualize the membrane domains, coated pits, and endocytic inclusions. Filipin, a probe for cholesterol, varied in density along the plasma membrane of secretory ameloblasts, and allowed us to visualize membrane remnants inside the forming enamel. With respect to phospholipids located in the extracellular matrix, the malachite-green-glutaraldehyde (MGA) method or iodoplatinate (IP) reaction retains and visualizes enamel and dentin phospholipids. In predentin, aggregates appearing as granules and filaments, or liposome-like structures, were located in the spaces between collagen fibrils. In dentin, organic envelopes coating the crystals, also named "crystal-ghost" structures, outlined groups of collagen fibrils. Histochemical data provided evidence that phospholipids are co-distributed or interact with proteoglycans. Radioautography after IP reaction established that [3H] choline was detected in dentin as early as 30 min after the intravenous injection of the labeled precursor, before any labeling was seen in odontoblasts and predentin. This suggests that blood-serum-labeled phospholipids pass between odontoblasts, cross the distal permeable junctional complex, and diffuse in dentin prior to any cellular uptake and phospholipid synthesis. Pharmacologically and genetically induced pathology also supports the suggestion that phospholipids play an important role in the formation and mineralization of dental tissues.